System for treatment and imaging using ultrasonic energy and microbubbles and related method thereof

ABSTRACT

A method and related system for providing therapy to a treatment site, such as stenosis or other vasculature disease, at one or more locations of a subject, such as the vasculature. The method includes: advancing an ultrasound catheter to or in proximity to the subject&#39;s treatment site; infusing microbubbles into or proximal to the treatment site; and delivering ultrasonic energy from the ultrasound catheter. The ultrasonic energy may be adapted for: imaging the treatment site, translating the microbubbles into or in the vicinity of the treatment site and/or rupturing the microbubbles.

RELATED APPLICATIONS

The present application is an application filed under 35 U.S.C. 371claiming priority to International Application PCT/US2008/081189, filedOct. 24, 2008, which claims priority to U.S. Provisional ApplicationSer. No. 61/000,632, filed Oct. 26, 2007, entitled “Molecular TargetedMicrobubbles for Enhanced Blood Vessel Imaging and Therapeutic Treatmentof Neointimal Hyperplasia;” and U.S. Provisional Application Ser. No.61/099,025, filed Sep. 22, 2008, entitled “Molecular TargetedMicrobubbles for Enhanced Blood Vessel Imaging and Therapeutic Treatmentof Neointimal Hyperplasia” Each of International ApplicationPCT/US2008/081189, U.S. Provisional Application Ser. No. 61/000,632 andU.S. Provisional Application Ser. No. 61/099,025 is hereby incorporatedherein, in its entirety, by reference thereto.

GOVERNMENT SUPPORT

This invention was made with government support under Federal Grant No.HL090700 and Federal Grant No. 5R01EB002185-7, awarded by The NationalInstitutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Cardiovascular disease (CVD) was blamed for 37% of the 2.4M deaths inthe US (2003) [1]. CVD is the leading cause of death in the US and thedeveloped world.

Currently Available Drug-Eluting Stents (DES) Pose a Major PotentialHealth Concern

The clinical use of Drug Eluting Stents (DES), in relation to Bare MetalStents (BMS), has evolved over a period of approximately 18 months fromapproximately 0% usage in the U.S., to the point where they were used inapproximately 80% of coronary stent procedures in the U.S. [2, 3].

The above cited recent studies indicates that there is a significant,growing population (approximately 6 Million individuals worldwide [4])who currently find themselves having been implanted with DES and face achoice between taking the expensive and risky drugclopidogrel—potentially for life—or increased risk of premature death.

The Vascular Smooth Muscle Cell, VCAM-1 and Rapamycin: Vascular SMCProliferation Contributes to Angioplasty-Induced Stenosis and In-StentRestenosis.

The primary function of the vascular SMC in adult animals is contractionand SMCs express a unique repertoire of genes that allow for thisspecialized form of contraction, including SM α-actin, smooth musclemyosin heavy chain (SMMHC), SM22α, calponin, desmin, smoothelin—genes werefer to as SMC differentiation marker genes [5-8]. This repertoire ofgenes is typically used to describe the “contractile” phenotype ormature SMC.

VCAM-1 is a Marker of the Phenotypically Modified/Proliferating SMC.

The changes in SMC gene expression profiles associated withinjury-induced phenotypic modulation are transient. That is, SMCsundergo phenotypic modulation as a natural response to repair theinjured blood vessel, transitioning from a contractile phenotype to asynthetic phenotype but revert back to a contractile phenotype as thelesion resolves itself. Thus, this continuum of altered SMC geneexpression profiles can be used to target the phenotypically modifiedSMC that invests in the developing neointima using molecular targeting.VCAM-1 (vascular cell adhesion molecule 1) is expressed in proliferatingSMCs [9, 10] and transiently upregulated in SMCs following acutevascular injury and in atherosclerotic lesions [11]. The function ofVCAM-1 is to promote cell-cell interaction required for SMC migrationand recruitment or attraction of other cell types into the lesion, e.g.VCAM-1 interaction on SMCs with integrins on leukocytes, monocytes ormacrophages (all inflammatory cells) [9]. Because VCAM-1 is expressed atmuch lower levels in the quiescent contractile SMC phenotype, butincreased in proliferating SMCs, VCAM-1 can thus be used to target theproliferating SMC.

Rapamycin is a Potent SMC Anti-Proliferative Agent and the Bench-MarkAgent for Preventing In-Stent Restenosis by Release from a DES.

The cell cycle consists of 5 basic steps: dormancy (G0) or thecontractile SMC phenotype, gap phase 1 (G1), synthesis (S), pre-mitosisor gap phase 2 (G2) and mitosis (M). In response to acute vascularinjury, SMCs leave G0 and enter G1 to begin the process of cellproliferation and division into M phase; this is the synthetic migratoryor proliferative SMC phenotype. The strategies for preventing SMCproliferation and entry into the cell cycle have been to block variousphases of the cell cycle once the cell has left G0 in response to injuryor some acute growth stimulus. Sirolimus, or rapamycin, and itsanalogues, ABT578 (Abbot Pharmaceuticals) and everolimus, areimmunosuppressants with both anti-inflammatory and antiproliferativeproperties that interfere early in the cell cycle by inhibiting thepassage of cells from G1 to S phase. Drugs that inhibit cell cycle inthe G1 phase are considered cytostatic and may be less toxic than drugsthat act later in the cell cycle [12, 13]. Rapamycin is the mostthoroughly investigated agent of this group and has become thebench-mark agent for the prevention of coronary artery restenosis [14].Thus, because rapamycin is considered “cytostatic”, SMCs treated withrapamycin do not die but maintain their viability in the growth arrestedstate.

Molecular Targeting of Microbubble Carriers

Recent research has investigated the feasibility of targeted ultrasoundcontrast microbubbles as a means of detecting intravascularmanifestations of disease. Pathology is often accompanied by alterationsof the endothelial cell layer lining of the affected blood vessels. Thisdysfunction may occur in the microcirculation, and is identified by theselective expression or up-regulation of certain molecules on thevascular endothelial surface. Many of the molecular markers ofendothelial dysfunction corresponding to disease states such asatherosclerosis [15], transplant rejection [16], inflammation andischemia reperfusion injury [17] are well characterized. However, thereis currently no non-invasive, clinically approved technique to assessthe extent and location of such vascular pathologies. Experimentalformulations of targeted microbubbles, which contain a surface-boundligand specific for the intended target, are injected intravascularlyand, after a short circulation period, are observed to accumulate at thetarget site. Subsequent ultrasound imaging enables determination of thelocation and extent of the targeted disease state [18]. This technique,known as “targeted contrast enhanced ultrasound”, may achieve highspatial resolution, real time imaging, and a linear or other measurablecorrelation between adherent microbubbles and the received signal.

There is therefore a need for, among other things, the drug, the drugcarrier, and the means of localizing delivery; and a means to guide thefocal delivery under real time image guidance.

SUMMARY OF THE INVENTION

There is a need for real time, noninvasive, imaging method to reliablyguide the focal delivery of antiproliferative drug to regions at risk ofrestenosis following angioplasty and/or stenting.

An aspect of some of the various embodiments of the invention comprisean ultrasound contrast agent that have a selected drug incorporated intothe bubble shell. In one embodiment, the drug may be rapamycin. Itshould be appreciated that the present invention is not limited to anyparticular drug or class of drug, or agent (or any other type of mediumor material being delivered to the location of the subject or thetreatment site or diagnostic site of the subject. An aspect of variousembodiments of the present invention may further comprise the means (atransducer [or transducer array] and its associated driving electronics)to deliver ultrasound energy (“therapeutic”) to break the bubbles insuch a manner as to focally deliver drug material to selected localcells. For example, but not limited thereto, the selected cells arethose on or in the wall of a selected blood vessel. The precisemechanisms and the optimal conditions for ultrasound mediated drugdelivery are heretofore not well understood. What is known fromextensive literature is that the combination of bubbles plus ultrasoundgreatly improve the delivery of drug (or gene) material through the cellmembrane. In an approach, the “therapeutic” ultrasound transducer isintimately integrated with an “imaging” ultrasound transducer thatprovides real-time, noninvasive imaging for guiding the precise deliveryof potent drugs to a selected tissue region. Similar transducers usedclinically are referred to as intravascular ultrasound (IVUS) catheters.Typically, the design of an optimal imaging transducer and an optimaltherapeutic transducer are different—e.g. the therapeutic transducer mayoperate in a high power transmit mode of about 0.5 to about 2 MHz,whereas the imaging transducer operates as a finely sampled imagingarray about 5 to about 30 MHz range. It should be appreciated that otherhigher and lower frequency modes may, of course, be employed within thecontext of the invention as desired or required. Nevertheless, it ispossible to make compromises in the transducer design and arrive at acommon design for both imaging and therapeutic effect.

The combined transducer may be catheter-based, maytransthoracically-based (i.e. “conventional” diagnostic ultrasound) andintravascularly, as is the case with IVUS—introduced through femoral orcarotid artery. The transducer may also be introduced via any natural orsynthetic body cavity/orifice (uretha, anus, vagina, mouth/esophagus orsurgical incision in any body part). Transducer designs (or aspectsthereof) for some of these applications or aspects of the applicationsmay be known in context of conventional diagnostic ultrasound and mostlarge vendors develop and market transducers for each of theseapplications.

The drug/contrast may be delivered systemically via intravenous (IV)injection or it may be delivered more locally such as from anaperture/conduit in a catheter placed into the veinous or arterialcirculatory system.

The drug may exist “side by side” with the agent—i.e. the drug not boundinto the bubble shell. When the drug is injected “side by side” it maybe dissolved in any suitable solvent appropriate for that drug (e.g.water, lipid, alcohol, or solid form—for example, in very fine particleform—like nanoparticle, etc. Moreover, the drug could be in a gas orsolid, for example, could be in the core or shell of the bubble(respectively)); in addition to being in the liquid phase, the drug maybe used in the solid dosage forms, such as in nanoparticle formulationsof kinds familiar to those skilled in the art.

The bubbles may be molecular targeted to enhance cell-specificselectivity—per the techniques, for example, described in the multiplepapers by Klibanov [19, 20] and colleagues.

An aspect(s) of various embodiments of the present invention may beprovide a number of novel and nonobvious features, elements andcharacteristics, such as but not limited thereto, the following:integrated image guidance of ultrasound-based local drug delivery;integrated image guidance of ultrasound-based local gene delivery;cell-specific molecular targeting of therapeutic agent; and ultrasoundimaging-based estimation of the delivery of therapeutic agent.

An aspect of an embodiment or partial embodiment of the presentinvention (or combinations of various embodiments in whole or in part ofthe present invention) comprises a method of providing therapy to atreatment site at one or more locations of a subject. The methodcomprising: advancing an ultrasound catheter to or in proximity to thesubject's treatment site, the catheter having a proximal region anddistal region; infusing microbubbles from the distal region of theultrasound catheter into or proximal to the treatment site; anddelivering ultrasonic energy from the distal region of the ultrasoundcatheter. The ultrasonic energy adapted for: imaging the treatment siteand rupturing the microbubbles. The ultrasonic energy may also adaptedfor translating the microbubbles.

An aspect of an embodiment or partial embodiment of the presentinvention (or combinations of various embodiments in whole or in part ofthe present invention) comprises an ultrasound catheter system forproviding therapy to a treatment site at one or more locations of asubject. The system comprising: a tubular member having a proximalregion and distal region, the proximal end of the ultrasound catheteradapted to advance to or in proximity to the subject's treatment site; amicrobubble reservoir in hydraulic communication with the tubularmember, the microbubble reservoir is adapted to release microbubblesthat are intended to be located into or proximal to the treatment site;an ultrasonic energy source in communication with the distal region ofthe tubular member. The ultrasonic energy adapted for: imaging thetreatment site and rupturing the microbubbles. The system furthercomprises a control circuitry configured to send electrical activationto the ultrasonic energy source. The ultrasonic energy may also adaptedfor translating the microbubbles.

A method and related system for providing therapy to a treatment site,such as stenosis or other vasculature disease, at one or more locationsof a subject, such as the vasculature. The method includes: advancing anultrasound catheter to or in proximity to the subject's treatment site;infusing microbubbles into or proximal to the treatment site; anddelivering ultrasonic energy from the ultrasound catheter. Theultrasonic energy may be adapted for: imaging the treatment site,translating the microbubbles into or in the vicinity of the treatmentsite and/or rupturing the microbubbles.

These and other objects, along with advantages and features of theinvention disclosed herein, will be made more apparent from thedescription, drawings and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the instant specification, illustrate several aspects and embodimentsof the present invention and, together with the description herein, andserve to explain the principles of the invention. The drawings areprovided only for the purpose of illustrating select embodiments of theinvention and are not to be construed as limiting the invention.

FIG. 1 provide a schematic illustration of an embodiment (or partialembodiment) of the present invention ultrasound catheter system 102 forproviding therapy (and/or diagnosis) to a treatment site at one or morelocations of a subject.

FIGS. 2(A)-(C) schematically illustrate various embodiments (or partialembodiments) of the present invention ultrasound catheter system forproviding therapy (and/or diagnosis) to a treatment site at one or morelocations of a subject.

FIGS. 3(A)-(C) schematically illustrate the arrays of the Forsbergarray, Bouakaz array, and present invention embodiment array,respectfully.

FIG. 4 schematically illustrate an embodiments (or partial embodiment)of the present invention ultrasound catheter system.

FIG. 5: illustrates the epifluorescence microscopy observations (FIGS.5A, B, C) and ultrasound backscatter imaging (FIGS. 5D, E, F) ofadherent microbubbles. Microcapillaries infused with buffer alone showno microbubble adhesion (A) and no ultrasound signal (dashed boxillustrates microcapillary location) (D). Few adherent microbubbles arevisible in flow-only microcapillaries (B), and the corresponding echo isidentifiable but weak. A large number of adherent microbubbles arepresent in a microcapillary exposed to radiation force at 122 kPa (C),and the corresponding echo is strong. Scale bar represents 5 μm.

FIG. 6 illustrates a 10 MHz (e.g., Sequoia CPS) image of mouse commoncarotid using microbubbles with dual targeting: polymeric sialyl LewisX(psLex) and anti-mouse VCAM-1. Cho et al. “Dual-Targeted Contrast” AHAAbstract 2006. See Weller G E, Villanueva F S, Tom E M, Wagner W R.Targeted ultrasound contrast agents: in vitro assessment of endothelialdysfunction and multi-targeting to ICAM-1 and sialyl Lewisx.

Biotechnol Bioeng. 2005 Dec. 20; 92(6):780-8, of which are herebyincorporated by reference herein.

FIG. 7 illustrates: at FIG. 7(A) a B-Mode of rat carotid (40 MHz, Vevo).Yellow arrows point to the blood vessel; at FIG. 7(B) a B-mode of a ratcarotid artery (12 MHz); and at FIG. 7(C) 10 MHz ultrasound imagingusing bubble sensitive/specific imaging mode. White tracing denotes thecarotid artery wall. White Scale bars=10 mm.

FIG. 8 illustrates prototype pulse echo responses of dual layer(multi-frequency) transducer. FIG. 8 illustrates: at FIG. 8(A) a lowfrequency layer pulse-echo response; at FIG. 8(B) an Experimental highfrequency pulse-echo response; at FIG. 8(C) an experimental highfrequency pulse echo response after inverse filtering; and at FIG. 8(D)an FEA simulation of proposed, improved (better acoustic matching) highfrequency layer design (without filtering) All plots are voltage echoresponse vs. time (μs)

FIG. 9 illustrates a diagram of targeted ultrasound contrastmicrobubble. The gas core is encapsulated by a lipid monolayer shell,which is coated with a PEG brush. The targeting ligand, here ananti-P-selectin monoclonal antibody, is secured to the distal tips ofthe polymers via a biotin-streptavidin link. Figure is not to scale.

FIG. 10 illustrates a microbubble adhesion at wall shear rate of 355 s-1on P-selectin after insonation at 122 kPa (122 kPa on P-sel; n=4),adhesion on P-selectin after flow alone (0 kPa on P-sel; n=3), andadhesion on casein after insonation at 122 kPa (122 kPa on Casein—i.e.control); n=3). Mean number of adherent microbubbles per 10 opticalfields+standard deviation. Insonated capillaries exhibited significantlygreater adhesion (p<0.05) than that of the flow only or insonatedcapillaries at each condition examined. The break in vertical scale maybe noted.

FIG. 11 illustrates an epifluorescence microscopy observations (FIGS.11(A), 11(B), 11(C)) and ultrasound backscatter imaging (FIGS. 8(D),8(E), 8(F)) of adherent microbubbles. Microcapillaries infused withbuffer alone show no microbubble adhesion (A) and no ultrasound signal(dashed box illustrates microcapillary location) (FIG. 11 (D)). Fewadherent microbubbles are visible in flow-only microcapillaries (FIG. 11(B)), and the corresponding echo is identifiable but weak. A largenumber of adherent microbubbles are present in a microcapillary exposedto radiation force at 122 kPa (FIG. 11 (C)), and the corresponding echois strong. Scale bar represents 5 μm.

FIGS. 12(A)-(B) schematically illustrate various embodiment (or partialembodiments thereof) of the present invention ultrasound cathetersystem.

FIG. 13 provides a plan schematic view of the microfluidic flow-focusingdevice or in-situ device.

FIGS. 14(A)-(B) provide a schematic elevation view of embodiments of thecatheter system having occlusion or sealing systems.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 provides a schematic illustration of an embodiment (or partialembodiment) of the present invention ultrasound catheter system 102 forproviding therapy to a treatment site at one or more locations of asubject. The system 102 may comprise a tubular member 118 such as acatheter or multiple catheters. The catheter(s) 118 having a proximalregion 115 and distal region 117, whereby the proximal end of theultrasound catheter is adapted or configured to be advanced to or inproximity to the subject's treatment site. It should be appreciated thatany one of the catheters as shown may be a plurality of catheters andany given catheter may have one or more lumens or channels therein. Thesystem further comprises a microbubble reservoir 132 in hydrauliccommunication with the tubular member. The microbubble reservoir is 132may be located in the proximal region 115 and/or the proximal region 117as desired or required. The microbubble reservoir is 132 may be adaptedto release microbubbles that are intended to be located into or proximalto the treatment site. The system further comprises an ultrasonic energy112 source in communication with the proximal region 115 and/or distalregion 117 of the tubular member 118. The ultrasonic energy 112 may becapable of: imaging the treatment site, and/or rupturing themicrobubbles. The ultrasonic energy 112 may be located outside or atleast partially surrounding the subject 113 or patient. The systemfurther comprises a control circuitry 100 or controller configured tosend electrical activation to the ultrasonic energy source 112 or anycomponents or subsystem affiliated with the catheter system 102.Further, the ultrasonic energy source 112 may provide ultrasonicradiation forces for translating the microbubbles into or in thevicinity of the treatment site; or alternatively the mechanical forcesmay be provided for translating the microbubbles into or in the vicinityof the treatment site, as well as a combination of both mechanical andultrasonic forces (acoustic wave) to achieve the desired or requiredresult.

The tubular member 118 and other components and subsystems affiliatedwith the catheter system 102 may be manufactured in accordance with avariety of techniques known to an ordinarily skilled artisan. Suitablematerials and dimensions can be readily selected based on the naturaland anatomical dimension of the treatment or diagnosis site and ondesired percutaneous access site or exterior.

For example, in an exemplary embodiment, the tubular body proximalregion 115 and/or distal region 117 comprises a material that hassufficient flexibility, kink resistance, rigidity and structural supportto push the ultrasound energy source 112 through the patient'svasculature or organ to a treatment site or vicinity thereof. Examplesof such materials include, but are not limited to, extrudedpolytetrafluoroethylene (“PTFE”), polyethylenes (“PE”), Pebax—made byArkema, polyamides and other similar materials. In certain embodiments,the tubular body proximal region 115 and/or distal region 117 isreinforced by braiding, mesh or other constructions to provide increasedkink resistance and ability to be pushed. For example, nickel titaniumor stainless steel wires can be placed along or incorporated into thetubular member or body 118 to reduce kinking. For example, variousguidewires, sheaths and additional tubular members may be implemented tohandle the communications, navigations, controlling and imaging, etc.

It should be appreciated that the aforementioned catheter device,reservoir, ultrasound, and controller may be disposed entirely insidethe applicable location of the subject as desired or required, outsidethe location of the subject as desired or required or a combination ofinside or outside the location of the subject. The one or more locationsof the subject may be an organ. The organ may include hollow organs,solid organs, parenchymal tissue, stromal tissue, and/or ducts. The oneor more locations of the subject may be a tubular anatomical structure.The tubular anatomical structure may be a blood vessel. Further, forexample, the treatment site may be a vasculature treatment sitecomprising at least one of the following: stenosis region or any regionexhibiting vascular disease.

In an approach, a manifold and/or axis port 114 couples severaltherapeutic and/or diagnostic devices typified by device 116 to thecatheter system 102. A syringe, flow-driver or pumping device 124 isalso in communication with the manifold 114. The catheter system 102 inturn may be delivered through a guide sheath 120 that may be incommunication with a navigation guide 122. In operation the physician oruser inserts one or more such catheter system 102 into the body of thesubject 113, for instance on going into the leg, chest or skull (orother anatomical part or parts or subject region or regions to cover thehollow or solid organs, blood vessels, etc.) under imaging guidance orother applicable examination or intervention. The same or similarultrasound visualization may be used to follow the progress of the oneor more implant(s) both acutely and chronically. This catheter devicemay have various interior and peripheral lumens, chambers and channels.Such interior and peripheral lumens, chambers and channels may be usedto deliver other devices and perform various diagnostic functions. Forexample, each lumen, chamber, and channel may communicate with aseparate port of the manifold 114. A lumen, chamber or channel maycontain a pressure transducer 128. Other lumens and channels may bedevoted to an optical or other type of cell counter device, for example,as shown generically as device 119 in FIG. 1. Such a device may operatewith two optical fibers (optical device or counter) located in twoseparate lumens and/or ports to measure the number of and viability ofcells, agents, drugs or microbubbles delivered by the catheter. Anexample of fiber optics related application/technology is discussed inU.S. patent application Ser. No. 10/444,884, filed May 23, 2003 (U.S.Application No. 2003/0204171, published Oct. 30, 2003), and of which arehereby incorporated by reference herein in their entirety.

It should be appreciated that many other embodiments of controller,catheter system, ultrasound energy source(s), manifold and/or axis port,proximal region, therapeutic and/or diagnostic devices, distal region,tubular member, other lumen(s), pressure transducer, microbubblereservoir, microbubble propeller or microbubble translator or propeller,flow channeling and recirculation means, microcoil means, pump means,pressure and flow-rate monitor means, imaging means, computer means,drug-eluting stents (DES), and other details of construction and useconstitute non-inventive variations of the novel and insightfulconceptual means, system, and technique which underlie the presentinvention. An example of systems and methods that may be implementedwith various embodiments of the present invention are provided in thefollowing commonly owned Applications: U.S. patent application Ser. No.10/444,884, filed May 23, 2003 (US Application No. 2003/0204171,published Oct. 30, 2003); PCT Application No. PCT/US2005/026738, filedJul. 28, 2005; and PCT Application No. 2006/005876, filed Feb. 16, 2006,and of which are hereby incorporated by reference herein in theirentirety.

It should be appreciated that as discussed herein, a subject may be ahuman or any animal. It should be appreciated that an animal may be avariety of any applicable type, including, but not limited thereto,mammal, veterinarian animal, livestock animal or pet type animal, etc.As an example, the animal may be a laboratory animal specificallyselected to have certain characteristics similar to human (e.g. rat,dog, pig, monkey), etc. It should be appreciated that the subject may beany applicable human patient, for example.

FIGS. 2(A)-(C) schematically illustrate various embodiments (or partialembodiments) of the present invention ultrasound catheter system forproviding therapy to a treatment site at one or more locations of asubject. The catheter system 202 may comprise a tubular member 218 suchas a catheter or multiple catheters. The catheter(s) having a proximalregion and distal region, whereby the proximal end of the ultrasoundcatheter is adapted or configured to be advanced to or in proximity tothe subject's treatment site. It should be appreciated that any one ofthe catheters 218 as shown may be a plurality of catheters and any givencatheter may have one or more lumens therein. The system furthercomprises a microbubble reservoir 232 in hydraulic communication withthe tubular member 218 and any lumens, channels, controllers orcommunication devices. The microbubble reservoir 232 is adapted torelease microbubbles that are intended to be located into or proximal tothe treatment site 210 at the desired or applicable location 211 of thesubject. The system 202 further comprises an ultrasonic energy source212 in communication with the distal region (or other region as desiredor required) of the tubular member 218 (or other components orsubsystems of the present invention). The ultrasonic energy is adaptedfor or capable of: imaging the treatment site 210, and rupturing themicrobubbles. The system 202 further comprises a control circuitry 200configured to send electrical activation to the ultrasonic energy source212, as well as other components and subsystems of the presentinvention. Further, the ultrasonic energy source 212 may provideultrasonic radiation forces for translating the microbubbles into or inthe vicinity of the treatment site 210 at the desired or applicablelocation 211 of the subject; or alternatively the mechanical forces maybe provided for translating the microbubbles into or in the vicinity ofthe treatment site 210, as well as a combination of both mechanical andultrasonic forces (acoustic wave) to achieve the desired or requiredresult.

It should be appreciated that the aforementioned catheter 218, reservoir232, ultrasound 212, and controller 200 may be disposed entirely insidethe applicable location of the subject, outside the location of thesubject or a combination of inside or outside the location of thesubject. The one or more locations 211 of the subject may be an organ.The organ may include hollow organs, solid organs, parenchymal tissue,stromal tissue, and/or ducts. The one or more locations 211 of thesubject may be a tubular anatomical structure. The tubular anatomicalstructure may be a blood vessel. Further, for example, the treatmentsite 210 may be a vasculature treatment site comprising at least on ofthe following: stenosis region or any region exhibiting vasculardisease. Further, for example, the treatment site 210 may be avasculature treatment site and/or a diagnostic site.

Development of Transducer/Instrumentation to Optimize Delivery of aTherapeutic Agent by Microbubble Carrier.

Spatially localized, focused, non-invasive/minimally invasive treatmentsrequire appropriate non invasive real time imaging to guide thelocalization of the therapeutic (focal) region with respect to selectedtarget site in the context of surrounding anatomy. This point may seemsimple but it has profound implications for non-invasive treatment. Thisparadigm further suggests attention be paid to ensuring that the focusedtreatment zone be accurately and reliably aligned with whatevernon-invasive imaging is used. The ideal model would be that the imageplane is coincident with the therapeutic point, line or plane.Frequently, a small imaging array is placed centrally within an aperture“cut out” from a larger therapeutic array. Rosenschein [21] describes a94 mm diameter therapeutic array into which a 7.5 MHz annular array isplaced in concentric fashion. The system was used successfully for invitro thrombolysis in bovine artery segments. Unger [22] describes (atleast conceptually) a transducer design incorporating therapeutic andimaging array elements with a common front face plane. In this example,the therapeutic array is placed within a hole in the imaging array. Alarge central “hole” in an array aperture gives rise to a near-fieldblind spot and distorted sidelobe patterns—typically grating loberelated due to the poor spatial sampling implicit by virtue of the“hole” in the aperture. Until now, much work has involved fixturing animaging array with respect to a therapeutic focused transducer/array[23-25]. An integrated imaging and therapeutic array, for example, wasdescribed by the University of Washington [26]. There is, however, noreason to believe that such an “integrated” array comprises exactlycoincident “therapeutic” and imaging arrays as proposed here. Theprecise need for defining a required level of “integration” is afunction of the particular application.

In the context of microbubble imaging, Bouakaz [27] has described a dualfrequency transducer (0.9 MHz and 2.8 MHz) array using interspersedelements. The element spacing is 0.5 mm—i.e. λ spacing at 2.8 MHz. Whenusing an interspersed element design it becomes doubly problematic toachieve adequate spatial sampling. Further, only <50% of potentialactive area for each array (in isolation) is available. This loss ofactive area limits maximal acoustic power delivery. Forsberg [28] hasalso described a multifrequency array in which three linear arrays (2.5MHz, 5 MHz and 10 MHz) were placed side-by-side with a common focalrange (50 mm). This approach works well within the one fixed focalregion but lacks the versatility to address other ranges.

In an aspect of an embodiment of the present invention, there may beprovided the imaging array immediately over the therapeutic array. Someadvantages of an embodiment of the present invention configuration maybe illustrated in FIG. 3. FIGS. 3(A)-(C) schematically illustrate thearrays of the Forsberg array (see FIG. 3(A)) having elevationalview—field intersection at one pre-selected range; Bouakaz array (seeFIG. 3(B)) with alternating elements of high and low frequency andhaving poor sampling and 50% area use per array; and an exemplarypresent invention embodiment of the stacked arrays (see FIG. 3(C))having fine sampling and 100% area usage. The transducer operatingfrequency may be inversely related to device thickness. The High and Lowfrequency transducer components denoted: HF and LF, respectively. Allthree transducers may be implemented with various embodiments of thepresent invention as desired or required.

FIG. 4 schematically illustrate an embodiment (or partial embodiment) ofthe present invention ultrasound catheter system 402 for providingtherapy (as well as diagnostic if desired or required) to a treatmentsite at one or more locations of a subject. The catheter system 402 maycomprise a tubular member such as a catheter body 418 such or multiplecatheters, needles, or lumens. The catheter(s) having a proximal regionand distal region, whereby the proximal end of the ultrasound catheteris adapted or configured to be advanced to or in proximity to thesubject's treatment site such as a stenotic risk region 410. It shouldbe appreciated that any one of the catheters 418 as shown may be aplurality of catheters and any given catheter may have one or morelumens therein. The system further comprises a microbubble reservoir,port or channel 433 in hydraulic communication with the tubular member418 and any lumens, channels, controllers or communication devicesrelated to the catheter system. The microbubble reservoir, port orchannel 433 is adapted to release microbubbles that are intended to belocated into or proximal to the treatment site 410 at the desired orapplicable location, such as a vessel or vessel wall 411 of the subject.The system 402 further comprises an ultrasonic energy source 412 incommunication with the distal region (or other region as desired orrequired) of the tubular member 418 (as well as other components orsubsystems of the present invention). The ultrasonic energy is adaptedfor, or capable of: imaging the treatment site 410, (some embodiments,for example, optionally pushing bubbles using ultrasound radiation force[29]), and rupturing the microbubbles. For instance, therapeutic array436 for bursting the microbubbles are provided (e.g., at low frequencyLF or as desired or required). Further, an imaging array 437 for imaging(e.g., at high frequency array HF or as desired or required).

Still referring to FIG. 4, the system 402 further comprise (although notshown) a control circuitry configured to send electrical activation tothe ultrasonic energy source, as well as other components and subsystemsof the present invention. Further, the ultrasonic energy source mayprovide ultrasonic radiation forces for translating the microbubbles 434into or in the vicinity of the treatment site 410 at the desired orapplicable location 411 of the subject; or alternatively the mechanicalforces may be provided for translating the microbubbles into or in thevicinity of the treatment site 410, as well as a combination of bothmechanical and ultrasonic forces (acoustic wave) to achieve the desiredor required result.

It should be appreciated that the aforementioned catheter 418,microbubble reservoir or channel 433, ultrasound source(s) 412, andcontroller may be disposed entirely inside the applicable location ofthe subject, outside the location of the subject or a combination ofinside or outside the location of the subject. The one or more locations411 of the subject may be an organ. The organ may include hollow organs,solid organs, parenchymal tissue, stromal tissue, and/or ducts. The oneor more locations 411 of the subject may be a tubular anatomicalstructure. The tubular anatomical structure may be a blood vessel.Further, for example, the treatment site 410 may be a vasculaturetreatment site comprising at least on of the following: stenosis regionor any region exhibiting vascular disease. Further, for example, thetreatment site 410 may be a vasculature treatment site and/or adiagnostic site.

As such, the approach illustrated in FIG. 4, provides, for instance, acatheter for delivery of drug loaded bubbles, ultrasound imaging ofbubbles/tissue, and ultrasound-based bubble destruction/drug delivery.

The imaging transducer/transducer array and the therapeutictransducer/transducer array may be identical. Whereas it is sometimesnecessary to optimize two transducers for two functions it is alsofeasible, if the transducer possesses sufficient performance versatility(e.g. high frequency bandwidth and high power capability) to use thesame transducer for both imaging and therapeutic function.

Ultrasound-Triggered Release of Rapamycin from Microbubbles AttenuatesSMC Proliferation Over 48 Hrs In Vitro.

As discussed above, the chemical and biological properties of rapamycinand why it is the benchmark reagent for preventing SMC proliferationassociated with vascular injury in vivo. This established the rationalefor choosing rapamycin for ultrasound-triggered microbubble carrierrelease. Multiple groups have shown that treatment of cultured SMCs withrapamycin reduces SMC proliferation [12, 30]. However, delivering ofrapamycin via ultrasound triggered release from a microbubble carrierhas not been performed.

Exemplary Design/Experiment

Ultrasound was applied to rat smooth muscle cells in conjunction withmodified ultrasound microbubbles containing rapamycin in their shells.The microbubbles were prepared by co-inventor A. L. Klibanov at UVA.Microbubbles were formed by self-assembly of a lipid monolayer duringthe ultrasonic dispersion of decafluorobutane gas in an aqueous micellarmixture of phosphatidylcholine (2 mg/ml) and Polyethylene Glycol (PEG)stearate (2 mg/ml) with rapamycin (0.2 mg/ml) and/or a trace amount of afluorescent dye DiI (Molecular Probes, Eugene, Oreg.), similarly to theprocedure described previously [31]. Fluorescently labeled DiImicrobubbles were used as a control to ensure that the microbubblevehicle alone did not cause an effect on the cells. The rapamycin drug,dissolved in 100% ethanol, was also used as a control with which tocompare the effect of the rapamycin microbubbles. We assured a strongadherence of cells to the OptiCell (Biocrystal, Westerville, Ohio)flasks by plating them with fibronectin for 24 hrs prior to plating anycells. Rat SMCs were plated at a low density and allowed to grow for 48hrs in DF10 media inside each of 12 OptiCells. Digital phase microscopylight images of the cells were taken at 5 hrs prior before treatment toestablish baseline conditions. All images were taken at 4×magnification. 24 hrs after plating, the media was replaced with freshmedia containing either the DiI microbubbles (vehicle control),rapamycin drug (drug control), or rapamycin microbubbles. Themicrobubbles (DiI or rapamycin) were added to the OptiCells at aconcentration of 10×10⁶ bubbles/ml and the rapamycin was added at aconcentration of 10 ng/ml. The microbubble concentrations were chosensuch that the number of microbubbles added contained an equivalentamount of rapamycin, ˜10 ng/ml. We ensured that the drug had an effecteven without prolonged exposure by taking half of the OptiCell flasksand giving them treatment for only two hours. After two hours thedrug/bubble-containing media was replaced with fresh media. The cells inthe OptiCell flasks received one of the following 6 treatments: DiIbubbles for 48 hours, rapamycin drug for 48 hours, rapamycin bubbles for48 hours, DiI bubbles for 2 hours, rapamycin drug for 2 hours, rapamycinbubbles for 2 hours. All conditions were tested in duplicate.

Following the placement of fresh media and microbubbles into eachOptiCell, ultrasound was applied to the entire area of cell growth. Oneat a time, each OptiCell was horizontally placed into a water bath (−37°C.). A focused 1 MHz (Panametrics, Waltham, Mass.) transducer wasimmersed in the water and located directly above the cells. A motioncontroller was used to traverse the transducer across the aperture ofthe OptiCell so as to evenly apply ultrasound to the entire area of cellgrowth. A 1 MHz, 35% BW, Gaussian pulse was applied at a PulseRepetition Frequency (PRF) of 1 kHz, 600 kPa peak, for the entireinsonation time (9 mins.). Images were taken at 4 locations within eachOptiCell. These locations were marked with a dot at the 5 hr time point.Subsequent images were taken at these same locations, 24 hours, and 48hours after treatment. The OptiCells were stored in a 37° C. incubator.

Results:

In FIGS. 5(A) and 5(B), we show that delivery of ˜10 ng/ml of rapamycinby ultrasound-triggered release form a microbubble carrier prevented SMCproliferation, depicted as a change in cell number, compared to releaseof a fluorescent membrane dye, DiI (Invitrogen), from an equivalentnumber of microbubble carriers. Moreover, quantitative analysis in FIG.5(D), shows that delivery of rapamycin (10 ng/ml) byultrasound-triggered release from a microbubble carrier was notdifferent from cells treated with free rapamycin drug (10 ng/ml) in thecell culture media. Similar results were observed in the set of 6OptiCells which were only treated for 2 hours post ultrasound and thenallowed to grow for 48 hrs (FIGS. 5(C) and 5(E)). Thus, these resultsshow (among other things) that rapamycin and an inert cell marking dye(DiI, FIG. 5(C)) can be delivered to SMCs by ultrasound-triggeredmicrobubble carrier release.

Next, non-invasive ultrasound imaging can play a critical role in theguidance of the therapeutic ultrasound that will localize the releaseand transcellular membrane delivery of the rapamycin drug. For instance,FIGS. 6 and 7 illustrate the current capabilities for fine-scalevisualization of rodent vasculature. FIG. 6 illustrates abubble-specific image of vessel wall-bound molecular-targeted(anti-VCAM-1) bubbles in the mouse common carotid artery (CCA) assessedusing 10 MHz bubble specific ultrasound imaging (e.g., Sequoia scanneror other commercial clinical ultrasound scanner). FIG. 7(A) is aVisualSonics VEVO 770 image of a rat carotid at 40 MHz demonstratingfine spatial resolution. FIGS. 7(B) and 7(C) are B-Mode, and contrastspecific, rat carotid images acquired at 12 MHz and 10 MHz, respectively(e.g., Sequoia scanner).

Transducer and Instrumentation

An exemplary transducer solution for dual function imaging therapeuticsis one in which the transducer elements are sufficiently versatile thatthey can accomplish both tasks—high frequency (HF) imaging and lowfrequency (LF) bubble manipulation/breaking. This enables a design inwhich the imaging plane and therapeutic planes are coincident.Deficiencies in these previous designs suggest the need for a superiorsolution.

The solution to the dual function requirement of the transducer (HF,high resolution, low intensity imaging and LF, high power bubblefracture) is to form a transducer with two active layers: one on top ofthe other (for example as shown in FIG. 3(C) or FIG. 4). Each layer isresonant at a widely disparate frequency—the lower one at about 1-2 MHzand the upper one at approximately 12 MHz. Conventional design wisdomrelating to stacked transducer layers would suggest that the twotransducer layers would cause high undesirable interference between theresonances associated with each of the layers. Nevertheless, ourexperience suggests that a two layer transducer will work provided thatthe transducer layers are well matched between each other and to thebacking material.

In an approach, a prototype dual layer single element transducer wasdesigned using 1:3 PZT/epoxy composite transducer layers. The acousticimpedance of each layer is approximately 15 MRayl. The backing is adense metal (tungsten) loaded epoxy with an acoustic impedance ofapproximately 9 MRayl. This transducer was fabricated to our design byVermon, Tours, France. The single element device, 1 cm in diameter andwith a focal depth of 5 cm, was constructed to test the viability of theproposed dual layer approach. FIG. 8(A) illustrates the pulse-echoresponses of the LF layer. The LF result exhibits a desired smooth,short duration, waveform. The high frequency layer in the currentprototype exhibits a reflection artifact that we attribute toreflections between the rear of the low frequency layer and the backingblock (FIG. 8(B)). In FIG. 8(C), we show that we can substantiallycorrect this deficiency by using an inverse filter designed to force theresponse to be more Gaussian (in the frequency domain). Alternatively,we will redesign and optimize the dual layer transducer using bettermatched transducer layers to minimize/eliminate internal reflections. Anearly FEA result for a modified design, using a better matched backing(i.e. 15 MRayl), is shown in FIG. 8(D).

The transducer may be designed for any one of many clinicalapplications. It may be for transcutaneous use and comprise aconventional phased or linear array (flat or curved, or contouredanatomically or ergonomically as desired or required). It may also bedesigned for transesophagael, transvaginal, transuretha, transrectal orintra-operative use. Examples of each of these form factor transducersare known in the field—usually comprising similar transducer structuresinside a plastic case adapted to the chosen anatomic use.

The transducer may also be formed in a catheter—as in intrasvascularultrasound (IVUS). IVUS catheters are currently widely marketed in theUS by Boston Scientific (Natick, Mass.) and Volcano (Rancho Cordova,Calif.). The Boston Scientific transducer typically comprises a singleelement that is rotated at high speed by a drive wire to form a coronalview. The transducer element in this transducer may be modified bychanging its operating frequency (i.e. lowered to around 2-15 MHz) tomake it suitable for breaking bubbles. The Volcano transducer isgenerally a circumferential phased array. Again, the frequency of thearray design may be modified (i.e. lowered to around 2-around 15 MHz) tomake it suitable for breaking bubbles. It is possible to potentially useeither a dual layer design—as described herein—or potentially use amodified design where a compromise between high frequency imaging andlow frequency bubble breaking is selected—e.g., instead of attempting tooperate imaging at about 25 MHz and breaking at about 2 MHz, a singlewideband design at about 15 MHz is capable of about 8 MHz breaking and20 MHz imaging. High bandwidth transducer design, such as by usingmultiple matching layers, for example, as known to those skilled in theart. As shown in FIGS. 4 and 12, for example, the catheter transducermay also include a continuous hollow port down which drug coatedcontrast is flushed during use. In this way, a stream of active contrastis emitted into the field of view of the transducer as shownschematically. (In clinical use, the blood flow in the coronaries is inthe “right” direction—i.e. blood flow is moving in the distalorientation.).

Notice also that other formats of drug media delivery are possible. Forexample: free dissolved (e.g. alcohol) rapamycin (or other drug) may betransferred side by side with plain contrast microbubbles down thehollow port. As indicated in Price's 1998 Circulation paper (“Deliveryof colloidal particles and red blood cells to tissue through microvesselruptures created by targeted microbubble destruction with ultrasound”Vol. 98, No 13, pp 1264-1267), local bubble breakage enables delivery ofcolloidal material (including potential drug in dissolved or undissolvedform) across microcirculatory vessel walls. Bubble could be injectedintravenously and dissolved rapamycin may be injected via the catheterport.

Normally lipid based bubbles are used. Other shell materials may beused—such as albumen-based or polymer-based shelled bubbles. Bubbleswith these shell materials are known in the field.

Instrumentation

Among various options available, the SonixRP (Ultrasonix, Richmond, BC,Canada) is a versatile platform to use as the base instrumentation forimplementing the invention. Of course, other scanner platforms may beprocured or designed/built as is well known to those in the field. TheRP, and its research capabilities (including high levelsoftware/hardware architecture), are described in detail in a recentpublication [32] [32].

It should be appreciated a number of marketed technology systems andcomponents may be implemented with the present invention such as by, butnot limited to, the following: the medical ultrasound companies include:Philips, Siemens, General Electric—also VisualSonics etc. However, itmay be noted that these are not catheter based companies.BostonScientific and Volcano are the primary IVUS companies.

In Vitro Radiation Force Enhanced Molecular Targeted Ultrasound

A problem encountered when using intravascular injected targetedcontrast agent is that, except in very small vessels, only a very smallfraction of the injected material will be sufficient close (<1 μm) tohave even a remote chance to form the intended molecular bond betweenligand and receptor. In vitro studies of targeted microbubble adhesionon substrates of P-selectin have reported that only a small percentageof the perfused microbubbles were specifically retained underphysiological flow conditions [e.g., Klibanov[33]]. Although detectionof single microbubbles is possible [34], low efficiency of microbubbletargeting requires a larger administered dose of microbubbles than wouldotherwise be required. Microbubbles exhibit rheological behavior similarto that of erythrocytes [35] and tend to migrate towards the center ofthe blood vessel. As most endothelial proteins extend only nanometers[36] from the endothelium, it is unlikely that many of the microbubblesflowing through the targeted vasculature come into contact with theintended molecular target. Microbubble attachment efficiency can beincreased by moving circulating microbubbles into contact with thevessel wall, thus increasing the frequency of microbubble: targetadhesive events. Dayton [37] and others [38] previously hypothesizedthat microbubble adhesion to the vascular endothelium may be enhanced byusing ultrasound radiation forces to propel freely flowing microbubblestowards the vessel wall. Adhesion of microbubbles [39] and acousticallyactive liposomes [40] under applied acoustic pressure in an avidin:biotin model system has been examined, and adhesion of targetedmicrobubbles to cultured endothelial cells has been reported [39].

Acoustic radiation traveling through a continuous media produces apressure gradient, which is experienced as a directional force bycompressible bubbles in the acoustic field. Two components of thisradiation force have been described: a primary force, which is directedaway from the source, and a secondary force, which is typicallyattractive between ultrasound contrast microbubbles [41]. The behaviorof single, free-stream microbubbles exposed to acoustic radiation haspreviously been examined rigorously [37, 41, 42]. Derivations of themagnitude of both primary and secondary forces in the linear range werepresented by Dayton [37], assuming a low duty factor, a constantmagnitude of pressure in each applied pulse, and a unidirectionalpressure gradient. The primary radiation force is proportional to thenegative time-averaged product of the microbubble volume and the spatialpressure gradient. For a microbubble driven at resonant frequency,assuming small-amplitude oscillations, the magnitude of the primaryradiation force is defined by

$\begin{matrix}{F_{1} = {\frac{2\pi\; P_{a}^{2}R}{\delta\;\omega_{0}\rho\; c}\left\lbrack \frac{D}{T} \right\rbrack}} & (1)\end{matrix}$where P_(a) is the peak applied acoustic pressure, R is the microbubbleresting radius, δ is the total damping coefficient, ρ is the mediumdensity, c is the velocity of sound in the bulk aqueous phase, and ω0 isthe microbubble resonant frequency. This term is scaled by D/T for apulsed field, where D is the pulse duration and 1/T is the pulserepetition frequency (PRF).

Targeting these microbubbles to P-selectin was achieved by conjugatingthe anti-P-selectin monoclonal antibody (mAb) Rb.40.34 [43] to thedistal tips of PEG chains via a streptavidin link, as shown in FIG. 9.The preparation of the targeted microbubbles used in this experiment hasbeen described in depth elsewhere [31, 44]. Trace amount (<1% of totallipid mass) of Da lipid dye (Molecular Probes, Eugene, Oreg.) was usedas a fluorescence probe for epi-illumination microscopy. Microbubbleswere conjugated to the targeting ligand the day of the experiment, andwere stored on ice in C4F10-saturated Dulbecco's Phosphate BufferedSaline Solution (DPBS) (Invitrogen, Carlsbad, Calif.). Microbubble sizedistribution and concentration was determined with a Coulter counter(Beckman-Coulter, Miami, Fla.).

A 2.25 MHz, 0.5″ diameter, 0.8″ focal depth ultrasound transducer(Panametrics V306, Waltham, Mass.) was used in this study. At a PulseRepetition Frequency (PRF) of 10 kHz, 40 sinusoidal cycles at afrequency of 2.0 MHz were applied. Microbubbles were insonated atacoustic pressures between 24.5 and 170 kPa. Upon cessation ofinsonation, 10 optical fields along a P-selectin coated microcapillarywithin the width of the applied ultrasound beam were observed andrecorded. Alternatively, some flow chambers were exposed to 2 minutes offlow alone, without insonation, in order to assess microbubble bindingin the absence of applied radiation force. The number of adherentmicrobubbles in each of 10 fields of view following insonation wasdetermined off-line. Microbubbles aggregates projecting normal to theoptical plane (downward into the flow stream) were counted as a singlebubble. Microbubble aggregation was assessed by counting the number ofcontiguous microbubbles adherent in the optical plane. Each flow chamberwas used for a single experiment. Statistical significance was testedwith a Student's t-test. We observed negligible binding of targetedmicrobubbles to casein-coated (i.e. control) microcapillaries both withand without the application of radiation force, see FIG. 10. We observeda statistically significant (p<0.05) increase in specific microbubbleadhesion to P-selectin due to applied radiation forces at each of themicrobubble concentrations examined. Applied radiation force increasedtargeted microbubble adhesion to P-selectin coated microcapillaries16-fold at 75.times.10.sup.6 B.sup.-ml.sup.-1 and over 60-fold at0.25.times.10.sup.6 B.sup.-ml.sup.-1 (or other sizes, volumes and rangesas required or desired).

Imaging of adherent microbubbles in flow chambers was also performedusing 14 MHz ultrasound imaging (e.g., on a Siemens Sequoia or similarclinical scanner). Microbubbles were infused into the flow chamber asdescribed above and exposed to 1 minute of flow alone at the indicatedshear rate, followed by one minute of insonation at 122 kPa or 1additional minute of flow only. It has also been determined thatmicrobubbles attached to the target substrate by acoustic radiationforce remain viable for ultrasound imaging. We observed no adherentmicrobubbles and received no ultrasound signal in microcapillariesinfused with buffer alone (FIG. 11(A), FIG. 11(D)). A contrast signal isvisible in FIG. 11(E), which shows an ultrasound image of amicrocapillary infused with 2.5×10⁶ B/ml for 2 minutes in the absence ofacoustic pressure then flushed with buffer. A representativefluorescence microscopy field of view in this capillary is presented inFIG. 11(C). FIG. 11(B). FIG. 11(C) shows a representative optical fieldof view from a microcapillary infused with 2.5×10⁶ bubbles/ml exposed to1 minute of flow only, 1 minute of insonation at 122 kPa and then salineflushed, in which extensive microbubble accumulation is evident. Thecorresponding echo shown in FIG. 11(F) is very strong. This suggeststhat the microbubbles targeted by means of radiation force at anacoustic pressure of 122 kPa remain intact and echogenic.

In summary, we have demonstrated some of the key components of some ofthe embodiments of the present invention method and system including,but not limited thereto, the following:

1. Rapamycin loaded microbubbles+ultrasound have a demonstrated,selective, anti-proliferative effect on rat SMCs.

2. VCAM-1, as well as other cell surface antigens including but notlimited to PECAM, is upregulated in proliferating SMCs in the rat andother animal models of stenosis and human restenosis.

3. Fine resolution ultrasound imaging can visualize vasculature anatomyand achieve high sensitivity/high specificity bubble imaging.

4. Dual frequency transducers for: a) high frequency imaging, and b) lowfrequency radiation force/bubble fracture.

5. Radiation force can be used to improve bubble molecular VCAM-1targeting attachment efficiency.

Related Exemplary Methods (and Related Systems)

Single Element Transducer (Typically Non Imaging Capable).

Our preliminary data provided promising early results using a simple,axisymmetrically focused, single element transducer. What is required isa dual function (low frequency bubble “busting” plus high frequencyimaging) transducer and associated instrumentation.

Transducer Array (Typically Imaging Capable).

An exemplary design may comprise 1:3 composite piezoceramic—epoxy activelayers stacked one over the other. (A “1:3” composite comprisespiezoelectric ceramic posts embedded in a polymer matrix—i.e. the twocomponents are electrically and mechanically in “parallel”. The 1:3configuration is the dominant composite configuration and is inwidespread commercial use.). The composite material possessesapproximate 50% ceramic volume fraction and possesses an acousticimpedance of approximately 15 MRayl. A dense, tungsten particle filled,backing block is used. A thin matching layer, approximately quarterwavelength matched for 12 MHz operation, is used over the top. Aconventional filled silicone rubber lens will be used to obtain anelevation focus. The elevational focal depth is approximately 15 mm.Specifically, we use approximately 12 MHz B-Mode imaging resulting in<200 μm lateral resolution and axial resolution. At this frequency,

is 125 μm. Consequently, for practical f#'s, (i.e. 1-2) a 200 μmresolution is feasible. An array system provides more than sufficientscanning frame rate (>100 frames/s for selected small fields ofview—e.g. 15 mm×15 mm). Focused ultrasound delivery is delivered at 1-2MHz. We are able to control the region over which a therapeutic effectis obtained to approximately 3λ—i.e. approximately a 2 mm spot size.

High Resolution, High Sensitivity, High Specificity, Bubble Imaging.

An objective is to provide anatomic B-Mode imaging capability, bubblespecific imaging and application of bubble fracture pulses under usercontrol. The anatomic B-Mode imaging are accomplished using standardB-Mode image formation techniques—i.e. optimized aperture apodization,fixed focus transmit, dynamic receive focusing, signal detection andscan conversion. Bubble specific imaging will be provided by using“Pulse Inversion” (PI)[45]—i.e. 1, −1 transmit polarity/amplitude;followed by “1”+“−1” processing to eliminate the linear component). Ifnecessary other bubble specific techniques such as amplitude scaling(i.e. 1,2 transmit polarity/amplitude; followed by (2×“1”)—“2”processing [46]) and the combination of PI and amplitude scaling (e.g.−1, 2, −1 transmit polarity/amplitude; followed by “−1”+“2”+“−1”processing [47]).

Low Frequency, Bubble Pushing and Bubble Destruction.

These modes use the low frequency elements of the array. The design ofan embodiment for the transducer comprises less than a total of 128elements (96, 12 MHz elements and 24, 2 MHz elements). In this way, bysimply reprogramming the selected transducer apertures from theavailable 128 transducer connector channels, we can switch betweenimaging and therapeutic modes of operation.

Design Rapamycin Microbubble Carrier System Capable ofUltrasound-Triggered Release

Bubble Making and Rapamycin Incorporation.

Microbubbles are prepared by self-assembly of a lipid monolayer duringthe ultrasonic dispersion of decafluorobutane gas in the aqueousmicellar mixture of phosphatidylcholine and PEG stearate (2 mg/ml) withrapamycin (0.2 mg/ml) and/or a trace amount of a fluorescent dye DiI(Molecular Probes, Eugene, Oreg.), similarly to the procedure describedearlier [48]. In some instances, membrane thickening is achieved byaddition of glycerol trioleate (thicker microbubble shell will harborincreased amounts of rapamycin) [49]. Free lipid, dye and rapamycin, notincorporated in the bubble shell is removed by sequential (3×)centrifugal flotation (100×g, 5 min), with the recycling of the firstwash to save reagents.

Rapamycin Quantitation.

Robust and sensitive high performance liquid chromatography (HPLC)procedures are described in the literature for clinical assays. We haveHPLC available in our laboratory and will implement such a procedure[50]. Briefly, the sample being tested (microbubbles or media) islyophilized and redissolved in chlorobutanol, centrifuged to removesediment; samples placed in the autosampler vials and HPLC performedwith UV detection against a calibration curve with a known amount ofrapamycin.

Rapamycin Release by Ultrasound: In Vitro Functional Bubble DestructionTesting.

An aqueous saline dispersion of rapamycin-containing microbubbles(10⁶-10⁷/ml particle concentration) will be placed in an OptiCell (USAScientific, Ocala, Fla.) in 10 ml volume. We will destroy bubbles byultrasound in the conditions described for the cell culture study,remove the microbubble particles from OptiCell and subject them tocentrifugal flotation to prove that residual microbubbles (if present)will be removed from the samples. We will then perform rapamycinquantitation in the bubble-free infranatant by HPLC technique asdescribed above.

Attachment of Anti-VCAM-1 Antibody to Microbubbles.

Coupling of anti-VCAM-1 antibody to microbubble surface is performed bystreptavidin coupling technique as described [44]. Briefly, during thepreparation of microbubbles, 2 mol % ofbiotin-PEG3400-phosphatidylethanolamine is added to the lipid mixture. Astreptavidin bridge technique is applied for biotinylated anti-VCAM-1antibody coupling to the microbubble surface as described earlier forother antibodies [33, 51]. Biotinylation of antibody molecule isperformed with biotin N-hydroxysuccinimide ester reagent at pH 7.5 inDPBS buffer. The degree of antibody biotinylation is tested using theHABA assay as described previously [e.g., Klibanov [33]]. By theadjustment of the antibody-to-biotin-NHS, an incubation ratio couplingof ˜1 biotin per antibody will be achieved. The ELISA test on VCAM-1antigen is used to confirm that biotinylation does not inactivate theantibody. Streptavidin-bubbles (10⁹/ml) are incubated with biotinylatedantibody on ice for 30 min; free antibody are removed from the bubblesby triple centrifugal flotation wash with degassed DPBS buffer in abucket-rotor centrifuge (100×g, 5 min). After repeated flotations, themean size of antibody-coated bubbles is normally ˜2.5 um, with >99% ofthe particles less than 8 um (particle size and concentration areevaluated with a Coulter Multisizer IIe instrument (Beckman Coulter,Miami, Fla.). The amount of attached antibody per bubble is tested byfluorescence spectroscopy labeling as described earlier; typically, ˜10⁵antibody molecules per microbubble are attached by this technique [51].

FIG. 12(A) schematically illustrate an embodiments (or partialembodiment) of the present invention ultrasound catheter system 1202 forproviding therapy (as well as diagnostic if desired or required) to atreatment site at one or more locations of a subject. The cathetersystem 1202 may comprise a tubular member 1218 or other conduit orchamber, such or multiple catheters, needles, or lumens. The catheter(s)having a proximal region and distal region, whereby the proximal end ofthe ultrasound catheter is adapted or configured to be advanced to or inproximity to the subject's treatment site or region 1210. It should beappreciated that any one of the tubular member 1218 as shown may be aplurality of tubular or conduit members and any given catheter or thelike may have one or more lumens therein. The system further comprises amicrobubble reservoir 1232 in hydraulic communication with the port orchannel 1233 and in hydraulic communication with the tubular member 1218and any lumens, channels, controllers or communication devices relatedto the catheter system. The microbubble reservoir 1232 and port orchannel 1233 is adapted to release microbubbles that are intended to belocated into or proximal to the treatment site 1210 at the desired orapplicable location 1211 of the subject 1211, such a vessel, organ,anatomical structure, anatomical tubular structure, or duct, etc. Thesystem 1202 further comprises an ultrasonic energy source(s) 1212 incommunication with the distal region (or other region as desired orrequired) of the tubular member 1218 (as well as other components orsubsystems or components of the present invention). The ultrasonicenergy is adapted for or capable of: imaging the treatment site 1210,and rupturing the microbubbles. For instance, therapeutic array 1236(comprising a predetermined ultrasound system design as desired orrequired) for bursting the microbubbles are provided (e.g., at lowfrequency LF or as desired or required). Further, an imaging array 1237(comprising a predetermined ultrasound system design as desired orrequired) is provided for imaging (e.g., at high frequency array HF oras desired or required). Further yet, the ultrasonic energy source 1238(comprising a predetermined ultrasound system design as desired orrequired) may provide ultrasonic radiation forces for translating ortransporting the microbubbles 1234 (e.g., at low frequency LF or highfrequency HF, or combination thereof, or as desired or required) into orin the vicinity of the treatment site 1210 or region at the desired orapplicable location 1211 of the subject.

Still referring to FIG. 12(A), the system 1202 further comprise(although not shown) a control circuitry configured to send electricalactivation to the ultrasonic energy source, as well as other componentsand subsystems of the present invention. Further, regarding thetranslation or transportation of the microbubbles or applicable medium,mechanical forces may be provided may be provided in place of theultrasound forces (acoustic wave) or in combination with the ultrasoundfor translating the microbubbles into or in the vicinity of thetreatment site 1210 to achieve the desired or required result.

It should be appreciated that the aforementioned catheter 1218,microbubble reservoir 1232, microbubble port or channel 1233, ultrasoundsource(s) 1212, and controller may be disposed entirely inside theapplicable location of the subject 1211, outside the location of thesubject or a combination of inside or outside the location of thesubject. The one or more locations 1211 of the subject may be an organ.The organ may include hollow organs, solid organs, parenchymal tissue,stromal tissue, and/or ducts. The one or more locations 1211 of thesubject may be a tubular anatomical structure. The tubular anatomicalstructure may be a blood vessel. Further, for example, the treatmentsite 1210 may be a vasculature treatment site comprising at least on ofthe following: stenosis region or any region exhibiting vasculardisease. Further, for example, the treatment site 1210 may be avasculature treatment site and/or a diagnostic site.

FIG. 12(B) schematically illustrate an embodiments (or partialembodiment) of the present invention ultrasound catheter system 1202 forproviding therapy (as well as diagnostic if desired or required) to atreatment site at one or more locations of a subject. The cathetersystem 1202 may comprise a tubular member such as a catheter body 1218such or multiple catheters, needles, conduits, housings, or lumens. Thecatheter(s) having a proximal region and distal region, whereby theproximal end of the ultrasound catheter is adapted or configured to beadvanced to or in proximity to the subject's treatment site or region1210. It should be appreciated that any one of the catheters 1218 asshown may be a plurality of catheters and any given catheter may haveone or more lumens therein. The system further comprises a microbubblereservoir 1232 in hydraulic communication with the port or channel 1233and in hydraulic communication with the tubular member 1218 and anylumens, channels, controllers or communication devices related to thecatheter system. The microbubble reservoir 1232 may be single usemicrobubble dose and high concentration. Moreover, the reservoir 1232may comprise multiple uses and have a variety of concentrations asdesired or required. The microbubble reservoir 1232 and/or port orchannel may be a capillary size or larger, or the microscale or smallersuch as a microchip, lab-on-a-chip, or in-situ design. The microbubblereservoir 1232 and port or channel 1233 is adapted to releasemicrobubbles that are intended to be located into or proximal to thetreatment site 1210 at the desired or applicable location, such as avessel or vessel wall 1211 of the subject. The system 1202 furthercomprises an ultrasonic energy source 1212 in communication with thedistal region (or other region as desired or required) of the tubularmember 1218 (as well as other components or subsystems of the presentinvention). The ultrasonic energy is adapted for or capable of: imagingthe treatment site 1210, and rupturing the microbubbles. For instance,therapeutic array 1236 for bursting the microbubbles are provided (e.g.,at low frequency LF or as desired or required). The therapeutic array1236 comprises a bubble rupture transducer that may be a rotating type;or may be a non-rotating type and be aligned with the radiation forcetransducer 1238 (or any combination thereof). Further, an imaging array1237 is provided for imaging (e.g., at high frequency array HF or asdesired or required). The imaging array 1237 may be rotating ornon-rotating and may be a single element or multiple element (or anycombination thereof). Further yet, the ultrasonic energy source 1238 mayprovide ultrasonic radiation forces for translating or transporting themicrobubbles 1234 (e.g., at low frequency LF or high frequency HF, orcombination thereof, or as desired or required) into or in the vicinityof the treatment site 1210 at the desired or applicable location 1211 ofthe subject. The radiation force transducer 1238 may be elongated andnon-rotating. Alternatively, the shape may also vary and it may rotateas well. Alternatively, rather than a radiation force transducer, ameans for transporting or translating may be implemented, such asmechanically or electrically. For instance, but not limited thereto,ejecting the bubbles with sufficient peripheral oriented velocity so asto translate quickly to the vessel wall.

Still referring to FIGS. 12(A)-(B), the system 1202 further comprise(although not shown) a control circuitry configured to send electricalactivation to the ultrasonic energy source, as well as other componentsand subsystems of the present invention. Further, regarding thetranslation or transportation of the microbubbles or applicable medium,mechanical forces may be provided in place of the ultrasound forces(acoustic wave) or in combination with the ultrasound for translatingthe microbubbles into or in the vicinity of the treatment site 1210 toachieve the desired or required result.

It should be appreciated that the aforementioned catheter 1218,microbubble reservoir 1232, microbubble port or channel 1233, ultrasoundsource(s) 1212, and controller may be disposed entirely inside theapplicable location of the subject, outside the location of the subjector a combination of inside or outside the location of the subject. Theone or more locations 1211 of the subject may be an organ. The organ mayinclude hollow organs, solid organs, parenchymal tissue, stromal tissue,and/or ducts. The one or more locations 1211 of the subject may be atubular anatomical structure. The tubular anatomical structure may be ablood vessel. Further, for example, the treatment site 1210 may be avasculature treatment site comprising at least on of the following:stenosis region or any region exhibiting vascular disease. Further, forexample, the treatment site 1210 may be a vasculature treatment siteand/or a diagnostic site.

Still referring to FIGS. 12(A)-(B), for example (as well as otherembodiments discussed herein), the system 1202 may comprise, but notlimited to the following:

-   -   Imaging transducer may be scanned single element or array;    -   Orientation of scanning transducer/array may be annular format        per conventional;    -   IVUS or may be longitudinal (or other) format;    -   Longitudinal format is like shown here for the radiation force        transducer and may be similar to the Siemens AcuNav intracardiac        catheter transducer array;    -   Radiation force transducer may be a single element, focused        element;    -   It might be an annular array for multiple focal option;    -   Frequency of each transducer/array may be different;    -   Radiation force transducer may be high frequency;    -   Imaging radiation may be high frequency;    -   Rupture radiation may be low frequency;    -   Rupture and imaging could be coincident—one over the other;    -   Bubbles are conceptually injected via a port;    -   Bubbles may be injected freely via the same access catheter        (i.e. ˜2 mm tube or as desired);    -   Bubbles may be saved in a single use highly concentrated from        near the catheter tip. This would allow us to use a smaller        number of bubbles. Keeping the bubbles in high concentration        (i.e. low rate of outward diffusion) allows them to be time        stable);    -   Bubbles may be monodisperse (all same size), but not        necessarily;    -   In principle, bubble dispersions can be sorted.

FIG. 13 illustrates a schematic plan view of the “On-chip generation ofmicrobubbles as a practical technology for manufacturing contrast agentsfor ultrasonic imaging” by Kanaka Hettiarachchi, Esra Talu, Marjorie L.Longo, Paul A. Dayton and Abraham R Lee Lab on a Chip, 2007, 7, 463-468.Provided is the soft molded PDMS (silicone) based micro flow chamberbelow. An aspect of the present invention may utilize some aspects. Anembodiment of the present invention provides a segment of a device thateasily fits at the tip of a catheter. This approach has many featuresand characteristics:

-   -   1. Increased versatility—can vary shell composition (i.e.        potentially drug/gene payload and concentration “on the fly”);    -   2. Enables otherwise unfeasible bubbles. Making the bubbles at        the tip means that stability problems are mitigated. The bubbles        only have to survive a few seconds before therapeutic delivery.        This may enable less stable chemical formulations or less stable        bubble (i.e. shell/gas) permutations. Currently, gas is limited        to one with very low rate of diffusion (i.e. high molecular        weight). The new design enables the use of new gases or light        gases at a minimum. This area isn't properly explored yet in our        opinion.    -   3. Existing problems with bubble stability that require complex        handling are circumvented.

Still referring FIG. 13, FIG. 13 provides a schematic plan view of themicrofluidic flow-focusing device or in-situ device. The microfluiddevice may be less than about 1 mm and therefore can be fit inside acatheter for example. The arrows indicate direction of flow of liquidinlet(s) and gas inlet.

It should be appreciated that the widths and heights may be larger orsmaller as required. The contours and shapes may vary as well.

FIG. 14(A) provides a schematic elevational view of an embodiment (orpartial embodiment) or approach of the present invention that provides asingle occlusion balloon to temporally stop flow—distal to transducerand drug bubble port. The balloon may be released (or partiallyreleased) after procedure (or during the procedure) and drug bubbleresidual or other medium flows systemically or as available.

FIG. 14(B) provides an embodiment similar to device as shown in FIG.14(A), however the instant embodiment or approach of the presentinvention provides a dual occlusion balloon to stop flow (or hinderflow) and create a sealed vessel section (or partially sealed section)in which drug (or applicable medium) is injected, delivered and thenflushed to eliminate systemic delivery concerns. The instant approachmay also include second port well separated from first so as to permitflush in from one and vacuum out at other—i.e. ports upstream anddownstream and close to each of the balloons (or located as desired orrequired).

The balloons may be any available sealing, occluding or blockingdesigns, structure, or devices available to those skilled in the art (orso as to provide partial occlusion when applicable or desired).

Examples of balloon (or occlusion) related catheter devices andassociated methods are provided below. The following patents,applications and publications as listed below are hereby incorporated byreference in their entirety herein. The devices, systems, and methods ofvarious embodiments of the invention disclosed herein may utilizeaspects disclosed in the following references, applications,publications and patents and which are hereby incorporated by referenceherein in their entirety:

-   -   1. U.S. Pat. No. 6,626,861, Sep. 30, 2003, “Balloon catheter        apparatus and method”, Hart, et al.    -   2. U.S. Patent Application Publication No. 2006/0235501, Oct.        19, 2006, “Stent supplying device”, Igaki, et al.    -   3. U.S. Patent Application Publication No. 2007/0055132, Mar. 8,        2007, “Catheter device,” Camus, et al.    -   4. U.S. Pat. No. 5,868,708, Feb. 9, 1999, “Balloon catheter        apparatus and method”, Hart, et al.    -   5. U.S. Patent Application Publication No. 2006/0189928, Aug.        24, 2006, “Catheter device”, Camus, et al.    -   6. U.S. Patent Application Publication No. 2008/0243233, Oct. 2,        2008, “Device and Methods for Treatment of Vascular        Bifurcations”, Ben-Muvhar, et al.    -   7. U.S. Pat. No. 5,222,970, Jun. 29, 1993, “Method of and system        for mounting a vascular occlusion balloon on a delivery        catheter”, Reeves, et al.    -   8. U.S. Pat. No. 5,707,354, Jan. 13, 1998, “Compliant catheter        lumen and methods”, Salmon, et al.    -   9. U.S. Patent Application Publication No. 2003/0163192, Aug.        28, 2003, “Methods for vascular reconstruction of diseased        arteries”, Wallace, et al.    -   10. U.S. Patent Application Publication No. 2002/0169496, Nov.        14, 2002, “Methods for vascular reconstruction of diseased        arteries”, Wallace, et al.    -   11. U.S. Patent Application Publication No. 2008/0103443, May 1,        2008, “Balloon catheter for treating hardened lesions”, Kabrick,        et al.    -   12. U.S. Pat. No. 6,565,601, May 20, 2003, “Methods for vascular        reconstruction of diseased arteries, Wallace, et al.    -   13. U.S. Pat. No. 5,827,171, Oct. 27, 1998, ‘Intravascular        circulatory assist device”, Dobak, et al.    -   14. U.S. Pat. No. 7,011,677, Mar. 14, 2006, “Methods for        vascular reconstruction of diseased arteries”, Wallace, et al.    -   15. U.S. Pat. No. 5,941,870, Aug. 24, 1999, “Catheter system        having a balloon angioplasty device disposed over a work element        lumen”, Jang, et al.    -   16. U.S. Patent Application Publication No. 2004/0158308, Aug.        12, 2004, “Delivery catheter for ribbon-type prosthesis and        methods of use”, Hogendijk, et al.    -   17. U.S. Patent Application Publication No. 2006/0161103,        “Catheter systems and methods for their use in the treatment of        calcified vascular occlusions”, Constantz, et al.    -   18. U.S. Patent Application Publication No. 2003/0199820, Oct.        23, 2003, “Catheter systems and methods for their use in the        treatment of calcified vascular occlusions”, Constantz, et al.    -   19. U.S. Patent Application Publication No. 2002/0044907, Apr.        18, 2002, “Catheter systems and methods for their use in the        treatment of calcified vascular occlusions”, Constantz, et al.    -   20. U.S. Patent Application Publication No. 2007/0049867,        “System for treating chronic total occlusion caused by lower        extremity arterial disease”, Shindelman, et al.    -   21. U.S. Pat. No. 5,041,089, Aug. 20, 1991, “Vascular dilation        catheter construction”, Mueller, et al.    -   22. U.S. Pat. No. 5,755,707, May 26, 1998, “Vascular dilating        catheter”, Miyagawa, et al.    -   23. U.S. Patent Application Publication No. 2004/0111145, Jun.        10, 2004, “Vascular prosthesis for the treatment of abdominal        aortic aneurysms, using a combined laparoscopic/open and        endovascular technique, and delivery system for releasing a        prosthesis fitted with anchoring stents”, Serino, et al.    -   24. U.S. Patent Application Publication No. 2007/0043389, Feb.        22, 2007, “System for treating chronic total occlusion caused by        lower extremity arterial disease”, Shindelman, et al.    -   25. U.S. Patent Application No. 2003/0220666, Nov. 27, 2003,        “Solid embolic material with variable expansion”, et al.    -   26. U.S. Pat. No. 5,117,831, Jun. 2, 1992, “Vascular catheter        having tandem imaging and dilatation components”, Jang, et al.    -   27. U.S. Pat. No. 6,527,979, Mar. 4, 2003, “Catheter systems and        methods for their use in the treatment of calcified vascular        occlusions”, Constantz, et al.    -   28. U.S. Pat. No. 5,447,503, Sep. 5, 1995, “Guiding catheter tip        having a tapered tip with an expandable lumen”, Miller, et al.    -   29. U.S. Pat. No. 7,198,637, Apr. 3, 2007, “Method and system        for stent retention using an adhesive”, Deshmukh, et al.    -   30. U.S. Pat. No. 5,415,634, May 16, 1995, “Catheter having        helical inflation lumen”, Glynn, et al.        Characteristics and Features that May be Implemented in Whole or        in Part (in any Permutation) with the Various Embodiments or        Partial Embodiments as Discussed Throughout this Document

An embodiment or approach of the present invention provides Dual useIVUS provides imaging plus therapy.

An embodiment or approach of the present invention provides Rapamycinbubbles (and other drugs with therapeutic effect—primarilyantiproliferative but could be others—including dual drug use—such asone drug to precondition tissue for a second drug to operate withefficacy).

Gene Bubbles

An embodiment or approach of the present invention provides the use ofcell-specific promoter constructs to target gene expression specificallyto one or multiple cell types in combination or independently. Thisincludes but is not limited to endothelial cell specific promoters (e.g.Tie-2, eNos), smooth muscle cell specific promoters (e.g. SMMHC, SMalpha-actin, SM22-alpha, myocardin), macrophages (e.g. mac-1) andpromoter of these genes that have been modified by mutating specific cisDNA sequences so as to limit inhibition of the promoter and increaseactivity. An example would be, but not limited, to a G/C mutation in theSM22a promoter which renders the promoter active in all smooth musclecell phenotypes [e.g., Wamhoff et al, Circ Res, 2004]. Genes undercontrol of a tissue selective promoter include but are not limited toanti-proliferative genes such p21, p53, KLF4 and proliferative genessuch as PCNA. In one scenario, a proliferative gene is targeted toendothelial cell to promote re-endothelialization and ananti-proliferative gene is targeted to smooth muscle to preventrestenosis.

An embodiment or approach of the present invention provides moleculartargeted bubbles (VCAM-1, PECAM, etc.). The targeting can be in contextof diagnosis or therapeutic use of bubbles—or both. The targeting to beany disease with molecular marker on endothelial surface. For example,VCAM-1 for atherosclerotic plaque—including “vulnerable plaque” or α_(ω) β₃ for angiogenesis associated with cancer.

An embodiment or approach of the present invention provides radiationforce and bubbles (which usually involves long pulse bursts, but notnecessarily).

An embodiment or approach of the present invention provides IVUScatheter with drug bubble delivery port upstream.

An embodiment or approach of the present invention provides drugdelivery “port” is plural and forms an annulus.

An embodiment or approach of the present invention provides amechanically scanned single element transducer—mechanically scanningachieves the regional coverage.

An embodiment or approach of the present invention provides phased arraytransducer—side fire/annular fire. The phased array may be used forimaging and therapy.

An embodiment or approach of the present invention provides acombination transducer elements—high power/low frequency, low power highfrequency.

An embodiment or approach of the present invention provides differenttransducer elements in different formats—e.g. phased array imaging plusscanned single element therapeutic.

An embodiment or approach of the present invention provides a singleocclusion balloon to temporally stop flow—distal to transducer and drugbubble port (for instance, release balloon after procedure and drugbubble residual flows systemically).

An embodiment or approach of the present invention provides a dualocclusion balloon to stop flow and create a sealed vessel section inwhich drug is injected, delivered and then flushed to eliminate systemicdelivery concerns (requires second port well separated from first so asto permit flush in from one and vacuum out at other—i.e. ports upstreamand downstream and close to each of the balloons)

An embodiment or approach of the present invention provides a 3Dscanning to record extent of problem lesion followed by automated 3Dsweep across the lesion to achieve therapeutic effect—i.e. it may betime/procedure efficient for the physician to outline the 3D extent ofthe plaque and then have the system sweep the region by way of automatedsequence of 1D lines to fully encompass the 2D surface of the 3D lesion.The “Track back” method, well known in IVUS, can be used “TrakBackII”from Volcano Corp for their array IVUS.

An embodiment or approach of the present invention provides a vulnerableplaque application as mentioned immediately above, except application isdiagnosis of vulnerable plaque. (Further, it doesn't actually doesn'thave to be 3D—but 3D is typically best). The means of differentiatingvulnerable plaque comprises-any permutation of:

-   -   a. Using appropriate molecular targeted microbubbles (VCAM-1 for        example).    -   b. Using microbubbles to detect microvasculature of vasa        vasorum—an indicator of active vulnerable plaque (see for        example, see reference Dutch group—Goertz, van der Steen et al.        http://publishing.eur.nl/ir/repub/asset/7950/060908_Frijlink,%20Martijn%20Egbert.pdf,        Harmonic Intravascular Ultrasound Thesis, Martijn Frijlink, 2006        Delft, Netherlands).    -   c. Performing signal processing (attenuation/frequency vs. depth        as per “virtual histology” of Volcano (Vince et al.)    -   d. Performing an elasticity based measurement to detect unusual        softness of plaque (e.g., per known methods of transducer inside        balloon described by M O'Donnell or measuring tissue response to        pulsatile blood forces—Van Der Steen)    -   e. “Tissue thermal strain imaging”: Identification of vulnerable        atherosclerotic plaque using IVUS-based thermal strain imaging:        Yan Shi; Witte, R. S.; O'Donnell, M.; Ultrasonics,        Ferroelectrics and Frequency Control, IEEE Transactions in        Volume 52, Issue 5, May 2005 Page(s):844-850

An embodiment or approach of the present invention sets forth tostabilize the vulnerable plague by delivery compounds such as basic FGFwhich promoter smooth muscle proliferation and migration to stabilizethe weak fibrous cap. We will refer to all analogous therapy approachesfor treating brain aneurysms with cerebral micro-coils. Micro-coils aredelivered to the blood vessel wall where an aneurysm occurred to providesupport for smooth muscle to proliferate and migrate and heal theaneurysm. An approach or embodiment promote, in the case, smooth muscleproliferation and migration, not inhibit it.

An embodiment or approach of the present invention provides atransducer(s) that may include any permutation of the following:

-   -   a. Single element capable of any or all of: radiation force,        imaging, bubble rupture.    -   b. Phased array (in any format: longitudinal or annular) capable        of any or all of: radiation force, imaging, bubble rupture.    -   c. Either or above wherein element(s) are dual (or triple) layer        arranged to provide (typically) high power at lower frequency        and lower power/fine resolution using high frequency.    -   d. Wherein the different transducers performing different        functions are not arranged one over the other. Place an        elongated radiation force transducer (or array) upstream of        imaging/delivery zone (see figure). Then have an imaging        transducer—imaging the bubbles that have been pushed to the zone        of interest. Then have a delivery transducer. (Subsets also        possible—such as dedicated elongated radiation force transducer        plus combined imaging/delivery transducer (or array).    -   e. An embodiment or approach of the present invention provides a        transducer(s) that can be formed from piezoelectric material        (preferably ceramic but could be piezoelectric polymer PVDF).        Alternatively transducers can be electrostatic, silicon (or        other material) “MEMS” devices.

An embodiment or approach of the present invention provides a method forlocalized delivery of drug from drug loaded microbubbles using highintensity ultrasound wherein the location of the focal delivery isguided by an integral, real-time, coincident, ultrasound imaging system.

An embodiment (or partial embodiment) or approach of the presentinvention provides a method for localized drug delivery wherein the drugcoated bubbles possess a selected molecular attachment ligand—such asVCAM-1, P-Selectin, etc. under realtime ultrasound image guidance, suchas:

-   -   dual targeting method—fast catch/slow hold    -   variant on bubbles such as liposomes    -   nanoparticle+bubble    -   dual modality contrast Ultrasound+MRI contrast Bubble+ferrous    -   potential of drug not being integrated in bubble shell but        existing in free solution aside the bubbles and relying on        bubble related sonoporation to result in preferential drug        uptake.

An embodiment or approach of the present invention provides a drug thatis rapamycin (antiproliferative, immunosuppressive, or antiinflammatorydrug, such as rapamycin, tacrolimus, paclitaxel, dexamethasone, or anactive analog or derivative, or combinations thereof). The drug may beselected from a group comprising actinomycin-D, batimistat, c-mycantisense, dexamethasone, paclitaxel, taxanes, sirolimus, tacrolimus andeverolimus, unfractionated heparin, low-molecular weight heparin,enoxaprin, bivalirudin, tyrosine kinase inhibitors, Gleevec, wortmannin,PDGF inhibitors, AG1295, rho kinase inhibitors, Y27632, calcium channelblockers, TRAM-34, IKCa channel blockers, amlodipine, nifedipine, andACE inhibitors, S1P1 and/or S1P3 receptor antagonists, sphingosinekinase 1 inhibitors, synthetic polysaccharides, ticlopinin,dipyridamole, clopidogrel, fondaparinux, streptokinase, urokinase,r-urokinase, r-prourokinase, rt-PA, APSAC, TNK-rt-PA, reteplase,alteplase, monteplase, lanoplase, pamiteplase, staphylokinase,abciximab, tirofiban, orbofiban, xemilofiban, sibrafiban, roxifiban, ananti-restenosis agent, an anti-thrombogenic agent, an antibiotic, ananti-platelet agent, an anti-clotting agent, an anti-inflammatory agent,an anti-neoplastic agent, a chelating agent, penicillamine, triethylenetetramine dihydrochloride, EDTA, DMSA (succimer), deferoxamine mesylate,a radiocontrast agent, a radio-isotope, a prodrug, antibody fragments,antibodies, gene therapy agents, viral vectors and plasmid DNA vectors.

An embodiment or approach of the present invention provides a subset ofrelevant bubble properties—dimensions, core gas, shell materials, etc.and including oily shell—decafluorobutane

An embodiment or approach of the present invention provides an acousticradiation force that may be used to translate bubbles towards a selectedvessel wall.

An embodiment or approach of the present invention provides microbubblesare targeted to blood vessels that routinely undergo and angioplastyand/or stenting (including balloon expansion stents and self-expandingstents), including but not limited to the coronary arteries, coronaryartery branch points, carotid arteries, cerebral arteries, femoralarteries.

An embodiment or approach of the present invention provides a systemicinjection of bubbles.

An embodiment or approach of the present invention provides a localizedinjection of bubbles—from catheter tip—preferably same catheter asimaging but potentially from separate one. See catheter cross-sectionaldrawing above.

An embodiment or approach of the present invention provides anultrasound image guidance of bubbles in a highly bubble-specific modeusing one of pulse inversion, amplitude scaling (“power modulation”) orcombination of two (“contrast pulse sequences”).

An embodiment or approach of the present invention provides anultrasound intensity has therapeutic (drug delivery) effect, whereinultrasound has cell death effect.

An embodiment or approach of the present invention provides the uses ofa ultrasound catheter—about 1-about 2 MHz therapeutic, about 30 MHzimaging.

An embodiment or approach of the present invention provides a co-locatedtransducer—imaging device overlaying the therapeutic device, imagingdevice residing in an aperture formed within center of therapeuticdevice (less desirable than overlaying).

An embodiment or approach of the present invention provides asynchronized operation—the imaging system is “gated” to never operatingduring the time of therapeutic operation.

An embodiment or approach of the present invention provides atherapeutic system “listens” for imaging system operation and insertstherapeutic pulses between imaging operations.

An embodiment or approach of the present invention provides an imagingsystem “listens” for therapeutic system operation and inserts imagingpulses between therapeutic operations.

An embodiment or approach of the present invention provides a “Pulsesequence” claims—X seconds (s) of therapeutic, followed by Y s ofimaging, and so on for Z minutes.

An embodiment or approach of the present invention provides anintegrating of this device on a catheter with other preferred catheterdevice options—e.g. balloon, pressure measurement, temperaturemeasurement, blood sampling.

An embodiment or approach of the present invention provides a catheterwith “over the wire” capability—the standard—has capability to be“threaded” over an in-place metal wire.

An embodiment or approach of the present invention provides a catheterthat may be a derivative of the “Volcano” IVUS catheter (phased annulararray). A therapeutic transducer—side firing—is placed near to theimaging annular array.

An embodiment or approach of the present invention provides a catheterthat may be related to some extent to the “Boston-Scientific” IVUScatheter (mechanically scanned single element) i.e. the existing highfrequency transducer element is replaced with a stack of low frequency(therapeutic) 1 MHz element with 30 MHz imaging overlaid. Alternatively,there are two transducers side by side in close proximity.

An embodiment or approach of the present invention provides a catheterpossessing an imaging transducer/array in any one or more of thefollowing formats: single element transducer rotated in circumferentialfashion to form coronal plane, circumferential array forming coronalplane, side-fire array and wherein the therapeutic array is in any oneof more of the following formats: single element transducer rotated incircumferential fashion to form coronal plane, circumferential arrayforming coronal plane, side-fire array

An embodiment or approach of the present invention provides an imagingtransducer/array is in any one or more of the following formats: singleelement transducer rotated in annular fashion to form coronal plane,annular array forming coronal plane, side-fire array and wherein thetherapeutic transducer is single focused element or annular array.

An embodiment or approach of the present invention provides apro-proliferative for filling up an aneurysm, occlusive treatmentupstream of an angiogenic region associated with evolving cancer; Imageguidance other than ultrasound; or other mechanisms for therapeuticdelivery—such as heat as opposed to acoustic disruption.

Wherein the image guidance (other than ultrasound) includes one or moreof: 1) X-ray and its derivatives (plain X-ray, realtime fluoroscopy andcomputed tomography [CT]), or 2) Magnetic Resonance Imaging (MRI)

An embodiment or approach of the present invention provides acomplementary drug operation—two drugs in different bubble populationsthat are stable in isolation but upon ultrasound disruption mix andbecome active/unstable/therapeutic.

An embodiment or approach of the present invention provides atherapeutic ultrasound plus bubble, drug and stent—wherein ultrasoundinduces vibrational mode/activity within stent so as to elicittherapeutic effect among cells/drugs/bubbles adjacent to stent surface.

An embodiment or approach of the present invention provides a differenttypes of stent and different generations of stent—bare metal stent,current DES, dissolving polymer stent, non polymer stent.

An embodiment or approach of the present invention provides an acousticsignature of stent that may be monitored to determine degree ofaccumulation of stiff acoustic loading on stent and any change resultingfrom therapeutic effect.

An embodiment or approach of the present invention provides microbubblesthat are delivered to a vascular aneurism to deliver a drug thatpromotes smoothmusclemgration and proliferation to heal the aneurism.Drugs include but are not limited to PDGF-BB, bFGF, etc.

An embodiment or approach of the present invention provides a method forlocalized drug delivery wherein the drug-carrying bubbles possess aselected molecular attachment ligand—such as VCAM-1, P-Selectin, etc.under realtime ultrasound image guidance including any permutationthereof:

-   -   dual targeting method—fast catch/slow hold [52]    -   3. microbubble composition, for use as in claims 1 and 2 such        that a plurality of targeting ligands capable of binding with        the diseased tissue, some of the ligands capable of binding        rapidly, and others binding firmly, are attached to the        microbubbles.    -   variant on bubbles such as liposomes    -   nanoparticle+bubble    -   Microbubble composition, such as in claim 1, 2 or 3, having        liposomes or biocompatible nanoparticles applied to the        microbubble shell to house the drug compounds to be released by        targeted insonation    -   dual modality contrast Ultrasound+MRI contrast Bubble+ferrous        (or in another disclosure)    -   potential of drug not being integrated in bubble shell but        existing in free solution aside the bubbles and relying on        bubble related sonoporation to result in preferential drug        uptake.

An embodiment or approach of the present invention provides a drug thatmay be rapamycin ((antiproliferative, immunosuppressive, orantiinflammatory drug, such as rapamycin, tacrolimus, paclitaxel,dexamethasone, or an active analog or derivative, or combinationsthereof).

An embodiment or approach of the present invention provides a subset ofrelevant bubble properties—dimensions, core gas, shell materials, etc.

An embodiment or approach of the present invention provides amicrobubble composition having drug incorporated, situated, dispersed,dissolved therein directly in the shell, core or core multiplicity, orattached to the outside of the shell, having shell(s) comprised withlipids, phospholipids, oils, fats, lipopolymers, polymers, proteins,surfactants or combinations thereof, shell thickness varied frommonomolecular 1 nm, to multimolecular and multilamellar, up to andincluding 1000 nm.

An embodiment or approach of the present invention provides microbubblecompositions having internal core filled with the gas, gas-vapor mixtureor gas precursor phase, gas having molecular mass from about 10 to about360.

An embodiment or approach of the present invention provides amicrobubble compositions having decafluorobutane core.

An embodiment or approach of the present invention provides an acousticradiation force is used to translate bubbles towards a selected vesselwall, or other organs or tissues as desired.

An embodiment or approach of the present invention provides anapplication in the coronary artery, application in other vessels, orother organs or tissues as desired.

An embodiment or approach of the present invention provides a systemicinjection of bubbles.

An embodiment or approach of the present invention provides a localizedinjection of bubbles—from catheter tip—preferably same catheter asimaging but potentially from separate one. See catheter cross-sectionaldrawing above.

An embodiment or approach of the present invention provides anultrasound image guidance of bubbles in a highly bubble-specific modeusing one of pulse inversion, amplitude scaling (“power modulation”) orcombination of two (“contrast pulse sequences”):

wherein ultrasound intensity has therapeutic (drug delivery) effect;and/or

wherein ultrasound has cell death effect.

An embodiment or approach of the present invention provides anultrasound catheter—1-2 MHz therapeutic, 30 MHz imaging.

An embodiment or approach of the present invention provides a co-locatedtransducer—imaging device overlaying the therapeutic device, imagingdevice residing in an aperture formed within center of therapeuticdevice (which may be less desirable than overlaying).

An embodiment or approach of the present invention provides asynchronized operation—the imaging system is “gated” to never operatingduring the time of therapeutic operation:

wherein the therapeutic system “listens” for imaging system operationand inserts therapeutic pulses between imaging operations, and/or

wherein the imaging system “listens” for therapeutic system operationand inserts imaging pulses between therapeutic operations.

An embodiment or approach of the present invention provides a “Pulsesequence” claims—X seconds (s) of therapeutic, followed by Y s ofimaging, and so on for Z minutes (time, repetition, cycles and durationas desired or required).

An embodiment or approach of the present invention provides anintegrating of this device on a catheter with other preferred catheterdevice options—e.g. balloon, pressure measurement, temperaturemeasurement, blood sampling.

An embodiment or approach of the present invention provides a catheterwith “over the wire” capability—the standard—has capability to be“threaded” over an in-place metal wire.

An embodiment or approach of the present invention provides a catheterthat is a derivative of the “Volcano” IVUS catheter (phased annulararray). A therapeutic transducer—side firing—is placed near to theimaging annular array.

An embodiment or approach of the present invention provides a catheterthat is a derivative of the “Boston-Scientific” IVUS catheter(mechanically scanned single element) i.e. the existing high frequencytransducer element is replaced with a stack of low frequency(therapeutic) about 1 MHz element with about 30 MHz imaging overlaid.Alternatively, there are two transducers side by side in closeproximity. Frequency may vary as desired or required.

REFERENCES CITED

The following patents, applications and publications as listed below arehereby incorporated by reference in their entirety herein. The devices,systems, and methods of various embodiments of the invention disclosedherein may utilize aspects disclosed in the following references,applications, publications and patents and which are hereby incorporatedby reference herein in their entirety:

-   -   1. U.S. Pat. No. 7,078,015, Unger, “Ultrasound Imaging and        Treatment”, Jul. 18, 2006.    -   2. U.S. Patent Application Publication No. 2005/017725 A1,        Hunter, William L., et. al., “Medical Implants and Anti-Scarring        Agents”, Aug. 11, 2005.    -   3. U.S. Patent Application Publication No. 2002/0082680 A1,        Shanley, John F., et. al., “Expandable Medical Device for        Delivery of Beneficial Agent”, Jun. 27, 2002.    -   4. U.S. Patent Application Publication No. 2003/0181973 A1,        Sahota, Harvinder, “Reduced Restinosis Drug Containing Stents”,        Sep. 25, 2003.    -   5. U.S. Patent Application Publication No. 2003/0206960 A1,        Iversen, Patrick L., et. al., “Delivery of        Microparticle-Conjugated Drugs for Inhibition of Stenosis”, Nov.        6, 2003.    -   6. U.S. Patent Application Publication No. 2003/0207907 A1,        Iversen, Patrick L., et. al., “Delivery of        Microparticle-Conjugated Drugs for Inhibition of Stenosis”, Nov.        6, 2003.    -   7. U.S. Patent Application Publication No. 2004/0077948 A1,        Violante, Michael R., “Echogenic Coatings with Overcoat”, Apr.        22, 2004.    -   8. U.S. Patent Application Publication No. 2004/0126400 A1,        Iverson, Patrick L., et. al., “Delivery of Therapeutic Compounds        Via Microparticles or Microbubbles”, Jul. 1, 2004.    -   9. U.S. Patent Application Publication No. 20040236414, Brar,        Balbir S., et. al., “Devices and Methods for Treatment of        Stenotic Regions”, Nov. 25, 2004.    -   10. U.S. Patent Application Publication No. 2004/0254635 A1,        Shanley, John F., et. al., “Expandable Medical Device for        Delivery of Beneficial Agent”, Dec. 16, 2004.    -   11. U.S. Patent Application Publication No. 2007/0010577 A1,        Lanza, Gregory, M., et. al., “Targeted Atherosclerosis        Treatment”, Jan. 11, 1007.    -   12. U.S. Patent Application Publication No. 2007/0003528 A1,        Consigny, Paul, et. al., “Intracoronary Device and Method of Use        Thereof”, Jan. 4, 2007.    -   13. U.S. Pat. No. 6,409,667, Hossack, et. al., “Medical        Diagnosis Ultrasound Transducer System and Method for Harmonic        Imaging”, Jun. 25, 2002.    -   14. U.S. Pat. No. 7,341,569 to Soltani, et al., “Treatment of        Vascular Occlusions Using Ultrasonic Energy and Microbubbles”,        Mar. 11, 2008.    -   15. U.S. Pat. No. 5,770,222 to Unger, et al., “Therapeutic Drug        Delivery Systems”, Jun. 23, 2008.    -   16. PCT International Application No. Serial No.        PCT/US2008/056643, filed Mar. 12, 2008, entitled, “Access Needle        Pressure Sensor Device and Method of Use,”    -   17. PCT International Application No. Serial No.        PCT/US2008/056816, filed Mar. 13, 2008, entitled, “Epicardial        Ablation Catheter and Method of Use,”    -   18. PCT International Application No. Serial No.        PCT/US2008/057626, filed Mar. 20, 2008, entitled, “Electrode        Catheter for Ablation Purposes and Related Method Thereof,”

REFERENCES CITED

The following patents, applications and publications as listed below andthroughout this document are hereby incorporated by reference in theirentirety herein. The devices, systems, and methods of variousembodiments of the invention disclosed herein may utilize aspectsdisclosed in the following references, applications, publications andpatents and which are hereby incorporated by reference herein in theirentirety:

LITERATURE CITED

-   1. Thom, T., et al., Heart disease and stroke statistics—2006    update: a report from the American Heart Association Statistics    Committee and Stroke Statistics Subcommittee. CIRCULATION, 2006.    113(6): p. e85-e151.-   2. Kandzari, D. E., et al., Frequency, Predictors, and Outcomes of    Drug-Eluting Stent Utilization in Patients With High-Risk    Non-ST-Segment Elevation Acute Coronary Syndromes. the American    Journal of Cardiology, 2005. 96(6): p. 750-755.-   3. Rao, S. V., et al., On-Versus Off-Label Use of Drug-Eluting    Coronary Stents in Clinical Practice (Report from the American    College of Cardiology National Cardiovascular Data Registry [NCDR]).    The American Journal of Cardiology, 2006. 97(10): p. 1478-1481.-   4. FDA, Circulatory Systems Devices Advisory Panel, 7-8 Dec. 2006.    Transcript:    http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfAdvisory/details.cfm?mtg=672,    2006.-   5. Hendrix, J., et al., 5′ CArG degeneracy in smooth muscle    {alpha}-actin is required for injury-induced gene suppression in    vivo. J. Clin. Invest., 2005. 115(2): p. 418-427.-   6. McDonald, O., et al., Control of SRF binding to CArG box    chromatin regulates smooth muscle gene expression in vivo. J. Clin.    Invest., 2006. 116(1): p. 36-48.-   7. Owens, G., M. Kumar, and B. Wamhoff, Molecular Regulation of    Vascular Smooth Muscle Cell Differentiation in Development and    Disease. Physiol. Rev., 2004. 84(3): p. 767-801.-   8. Wamhoff, B., et al., L-type Voltage-Gated Ca2+ Channels Modulate    Expression of Smooth Muscle Differentiation Marker Genes via a Rho    Kinase/Myocardin/SRF-Dependent Mechanism. Circulation    Research, 2004. 95(4): p. 406-414.-   9. Braun, M., et al., Cellular adhesion molecules on vascular smooth    muscle cells. Cardiovascular Research, 1999. 41(2): p. 395-401.-   10. Braun-Dullaeus, R., et al., Cell cycle-dependent regulation of    smooth muscle cell activation. Arterioscler Thromb Vasc Biol, 2004.    24: 845-850, 2004: p. 845-850.-   11. Landry, D., et al., Activation of the NF-kappa B and I kappa B    system in smooth muscle cells after rat arterial injury. Induction    of vascular cell adhesion molecule-1 and monocyte chemoattractant    protein-1. Am J Pathol, 1997. 151(4): p. 1085-1095.-   12. Parry, T., et al., Drug-eluting stents: sirolimus and paclitaxel    differentially affect cultured cells and injured arteries. Eur J    Pharmacol, 2005. 524(1-3): p. 19-29.-   13. Wessely, R., A. Schomig, and A. Kastrati, Sirolimus and    Paclitaxel on Polymer-Based Drug-Eluting Stents: Similar But    Different. Journal of the American College of Cardiology, 2006.    47(4): p. 708-714.-   14. Webster, A., et al., Target of rapamycin inhibitors (sirolimus    and everolimus) for primary immunosuppression of kidney transplant    recipients: a systematic review and meta-analysis of randomized    trials. Transplantation, 2006. 81(9): p. 1234-1248.-   15. Ross, R., The pathogenesis of atherosclerosis: a perspective for    the 1990s. Nature, 1993. 362: p. 801-809.-   16. Denger, T. and T. Pober, Cellular and molecular biology of    cardiac transplant rejection. Journal of Nuclear Cardiology, 2000.    7: p. 669-685.-   17. Sheridan, F., P. Cole, and D. Ramage, Leukocyte adhesion to the    coronarymicrovasculature during ischemia and reperfusion in an in    vivo canine model. CIRCULATION, 1996. 93: p. 1784-1787.-   18. Villanueva, F., A. Klibanov, and W. Wagner,    Microbubble-endothelial cell interactions as a basis for assessing    endothelial function. ECHOCARDIOGRAPHY, 2002. 19: p. 427-438.-   19. Klibanov, A. L., Targeted Delivery of Gas-Filled Microspheres,    Contrast Agents for Ultrasound Imaging. Advanced Drug Delivery    Reviews, 1999. 37: p. 139-157.-   20. Klibanov, A., et al., Targeted ultrasound contrast agent for    molecular imaging of inflammation in high-shear flow. Contrast Media    and Molecular Imaging, 2006. 1(6): p. 259-266.-   21. Rosenschein, U., et al., Ultrasound Imaging-Guided Noninvasive    Ultrasound Thrombolysis. CIRCULATION, 2000. 102: p. 238.-   22. Unger, E. and D. Yellohair, Methods and apparatus for performing    diagnostic and therapeutic ultrasound simultaneously U.S. Pat. No.    5,558,092, 1996.-   23. Chan, An image-guided high intensity focused ultrasound device    for uterine fibroids treatment. Medical Physics, 2002. 29(11): p.    2611-20.-   24. Vaezy, S., et al., Ultrasound image-guided therapy. Academic    Radiology, 2003. 10(8): p. 956.-   25. Vaezy, S., et al., High intensity focused ultrasound for    hemostasis of femoral artery catheter wounds. Ultrasound in Medicine    and Biology, 2006. 32(5 Supplement 1): p. 100.-   26. Crum, L., Guided High Intensity Focused Ultrasound (HIFU) for    Mission-Critical Care.    http://www.nsbri.org/Research/Projects/viewsummary.epl?pid=133,    2004.-   27. Bouakaz, A., F. Cate, and N. de Jong, A new ultrasonic    transducer for improved contrast nonlinear imaging. Physics in    Medicine & Biology, 2004. 49(16): p. 3515-3525.-   28. Forsberg, F., et al., Design and acoustic characterization of a    multi-frequency harmonic array for nonlinear contrast imaging.    Proceeding of 2001 IEEE Ultrasonics Symposium, 2001. 2: p.    1721-1724.-   29. Rychak, J., A. Klibanov, and J. Hossack, Acoustic Radiation    Force Enhances Targeted Delivery of Ultrasound Contrast    Microbubbles: In vitro Verification. IEEE Transactions on    Ultrasonics Ferroelectrics & Frequency Control, 2005. 52(3): p.    421-433.-   30. Marx, S., et al., Rapamycin-FKBP Inhibits Cell Cycle Regulators    of Proliferation in Vascular Smooth Muscle Cells. Circulation    Research, 1995. 76(3): p. 412-417.-   31. Klibanov, A., et al., Attachment of ligands to gas-filled    microbubbles via PEG spacer and lipid residues anchored at the    interface. Proc. Intl. Symp. Control. Rel. Bioact. Mat., 1999.    26: p. 124-125.-   32. Wilson, T., et al., The ultrasonix 500RP: A commercial    ultrasound research interface. IEEE Transactions Ultrasonics,    Ferroelectrics and Frequency Control, 2006. 53(10): p. 1772-1782.-   33. Takalkar, A., et al., Binding and detachment dynamics of    microbubbles targeted to P-selectin under controlled shear flow.    Journal of Controlled Release, 2004. 96(3): p. 473-482.-   34. Klibanov, A., et al., Detection of individual microbubbles of an    ultrasound contrast agent: fundamental and pulse inversion imaging.    Academic Radiology, 2002: p. S279-S281.-   35. Jayaweera, A., et al., In vivo myocardial kinetics of air-filled    albumin microbubbles during myocardial contrast echocardiography.    Comparison with radiolabeled red blood cells. Circulation    Research, 1994. 74(6): p. 1157-1165.-   36. Springer, T., Adhesion receptors of the immune system.    Nature, 1990. 347: p. 425-434.-   37. Dayton, P., et al., Acoustic radiation force in vivo: a    mechanism to assist targeting of microbubbles. Ultrasound in    Medicine & Biology, 1999. 25(8): p. 1195-1201.-   38. Fowlkes, J., et al., The role of acoustic radiation force in    contrast enhancement techniques using bubble-based ultrasound    contrast agents. Journal of the Acoustical Society of America, 1993.    93: p. 2348.-   39. Zhao, S., et al., Radiation force assisted targeting facilitates    ultrasonic molecular imaging. Molecular Imaging, 2004. 3: p. 1-14.-   40. Shortencarier, J., et al., A method for radiation-force    localized drug delivery using gas-filled lipospheres. IEEE Trans.    Ultrasonics, Ferroelectrics and Frequency Control, 2004. 51: p.    822-831.-   41. Dayton, P., et al., A preliminary evaluation of the effects of    primary and secondary radiation forces on acoustic contrast agents.    IEEE Transactions on Ultrasonics Ferroelectrics & Frequency    Control, 1997. 44(6): p. 1264-1277.-   42. Dayton, P., J. Allen, and K. Ferrara, The magnitude of radiation    force on ultrasound contrast agents. Journal of the Acoustical    Society of America, 2002. 112: p. 2183-2192.-   43. Bosse, R. and D. Vestweber, Only simultaneous blocking of the    L-and P-selectin completely inhibits neutrophil migration into mouse    peritoneum. European Journal of Immunology, 1994. 24: p. 3019-3024.-   44. Lindner, J., et al., Ultrasound Assessment of Inflammation and    Renal Tissue Injury With Microbubbles Targeted to P-Selectin.    CIRCULATION, 2001. 104(17): p. 2107-2112.-   45. Burns, P., S. Wilson, and D. Simpson, Pulse inversion imaging of    liver blood flow: improved method for characterizing focal masses    with microbubble contrast. Invest Radiol., 2000. 35(1): p. 71.-   46. BrockFisher, G. A., M.D. Poland, and P. G. Rafter, Means for    increasing sensitivity in non-linear ultrasound imaging systems U.S.    Pat. No. 5,577,505, 1996.-   47. Phillips, P., Contrast Pulse Sequences (CPS): Imaging non-linear    microbubbles. Proceedings of the 2001 IEEE Ultrasonics    Symposium, 2001. 2: p. 1739-1745.-   48. Klibanov, A., et al., Proceedings of 26th International    Symposium on Controlled Release of Bioactive Materials, Boston.    Controlled Release Society, 1999: p. 124-125.-   49. Unger, E., et al., Acoustically active lipospheres containing    paclitaxel—A new therapeutic ultrasound contrast agent.    Investigative Radiology, 1998. 33: p. 886-892.-   50. Boudennaia, T. Y. and K. L. Napoli, Validation of a practical    liquid chromatography with ultraviolet detection method for    quantification of whole-blood everolimus in a clinical TDM    laboratory. Therapeutic Drug Monitoring, 2005. 27(2): p. 171-177.-   51. Lindner, J. R., et al., Ultrasound assessment of inflammation    and renal tissue injury with microbubbles targeted to P-selectin.    Circulation, 2001. 104(17): p. 2107-2112.-   52. Klibanov, A., et al., Polymeric sialyl Lewis X microbubbles:    targeted ultrasound contrast agents for molecular imaging of    inflammation. RSNA Abstract Book, 2006 (Abs. # SSK06-06): p. 436-7.

It should be appreciated that various sizes, dimensions, contours,rigidity, shapes, flexibility and materials of any of the embodimentsdiscussed throughout may be varied and utilized as desired or required

It should be appreciated that the related components and subsystemsdiscussed herein may can take on all shapes along the entire continualgeometric spectrum of manipulation of x, y and z planes to provide andmeet the anatomical and structural demands and requirements.

Unless clearly specified to the contrary, there is no requirement forany particular described or illustrated activity or element, anyparticular sequence or such activities, any particular size, speed,material, duration, contour, dimension or frequency, or any particularlyinterrelationship of such elements. Moreover, any activity can berepeated, any activity can be performed by multiple entities, and/or anyelement can be duplicated. Further, any activity or element can beexcluded, the sequence of activities can vary, and/or theinterrelationship of elements can vary. It should be appreciated thataspects of the present invention may have a variety of sizes, contours,shapes, compositions and materials as desired or required.

In summary, while the present invention has been described with respectto specific embodiments, many modifications, variations, alterations,substitutions, and equivalents will be apparent to those skilled in theart. The present invention is not to be limited in scope by the specificembodiment described herein. Indeed, various modifications of thepresent invention, in addition to those described herein, will beapparent to those of skill in the art from the foregoing description andaccompanying drawings. Accordingly, the invention is to be considered aslimited only by the spirit and scope of the following claims, includingall modifications and equivalents.

Still other embodiments will become readily apparent to those skilled inthis art from reading the above-recited detailed description anddrawings of certain exemplary embodiments. It should be understood thatnumerous variations, modifications, and additional embodiments arepossible, and accordingly, all such variations, modifications, andembodiments are to be regarded as being within the spirit and scope ofthis application. For example, regardless of the content of any portion(e.g., title, field, background, summary, abstract, drawing figure,etc.) of this application, unless clearly specified to the contrary,there is no requirement for the inclusion in any claim herein or of anyapplication claiming priority hereto of any particular described orillustrated activity or element, any particular sequence of suchactivities, or any particular interrelationship of such elements.Moreover, any activity can be repeated, any activity can be performed bymultiple entities, and/or any element can be duplicated. Further, anyactivity or element can be excluded, the sequence of activities canvary, and/or the interrelationship of elements can vary. Unless clearlyspecified to the contrary, there is no requirement for any particulardescribed or illustrated activity or element, any particular sequence orsuch activities, any particular size, speed, material, dimension orfrequency, or any particularly interrelationship of such elements.Accordingly, the descriptions and drawings are to be regarded asillustrative in nature, and not as restrictive. Moreover, when anynumber or range is described herein, unless clearly stated otherwise,that number or range is approximate. When any range is described herein,unless clearly stated otherwise, that range includes all values thereinand all sub ranges therein. Any information in any material (e.g., aUnited States/foreign patent, United States/foreign patent application,book, article, etc.) that has been incorporated by reference herein, isonly incorporated by reference to the extent that no conflict existsbetween such information and the other statements and drawings set forthherein. In the event of such conflict, including a conflict that wouldrender invalid any claim herein or seeking priority hereto, then anysuch conflicting information in such incorporated by reference materialis specifically not incorporated by reference herein.

We claim:
 1. A method of providing therapy to a treatment site at one ormore locations of a subject, said method comprising: advancing anultrasound catheter to or in proximity to the subject's treatment site,said catheter having a proximal region and distal region; infusingmicrobubbles from said distal region of said ultrasound catheter into orproximal to the treatment site; delivering therapeutic ultrasonic energyfrom within said distal region of said ultrasound catheter to saidmicrobubbles to focally deliver a therapeutic material to the treatmentsite; and delivering imaging ultrasonic energy from within said distalregion of said ultrasound catheter to the treatment site to provide realtime imaging for guiding precise delivery of said therapeutic materialto the treatment site.
 2. The method of claim 1, further comprising:controlling activation of said therapeutic ultrasonic energy.
 3. Themethod of claim 1 wherein said one or more locations comprise at least aportion of an organ.
 4. The method of claim 3, wherein said organcomprises hollow organs, solid organs, parenchymal tissue, stromaltissue, and/or ducts.
 5. The method of claim 1, where the one or morelocations of the subject comprises at least a portion of a tubularstructure.
 6. The method of claim 5, wherein said tubular structurecomprises a blood vessel.
 7. The method of claim 6, wherein thetreatment site is a vasculature treatment site comprising at least oneof the following: stenosis region or any region exhibiting vasculardisease.
 8. The method of claim 1, wherein said delivering imagingultrasonic energy comprises delivering high frequency ultrasoundtransmission and reception.
 9. The method of claim 8, wherein said highfrequency comprises a range of about 2 MHz to about 50 MHz.
 10. Themethod of claim 8, wherein said high frequency comprises a range ofabout 5 MHz to about 30 MHz.
 11. The method of claim 8, wherein saidhigh frequency comprises a range of about 12 MHz to about 50 MHz. 12.The method of claim 8, wherein said high frequency comprises a range ofabout 12 MHz to about 30 MHz.
 13. The method of claim 8, wherein saidhigh frequency comprises about 20 MHz.
 14. The method of claim 8,wherein said high frequency comprises about 25 MHz.
 15. The method ofclaim 8, wherein said high frequency comprises about 30 MHz.
 16. Themethod of claim 1, wherein said delivering therapeutic ultrasonic energyfrom said distal region of said ultrasound catheter to said microbubblesto focally deliver a therapeutic material to the treatment sitecomprises delivering low frequency ultrasound energy to saidmicrobubbles and rupturing at least a portion of said microbubbles. 17.The method of claim 16, wherein said delivering therapeutic ultrasonicenergy from said distal region of said ultrasound catheter to saidmicrobubbles to focally deliver a therapeutic material to the treatmentsite comprises delivering high power ultrasound energy to saidmicrobubbles and rupturing at least a portion of said microbubbles. 18.The method of claim 17, wherein said high power delivering of ultrasonicenergy generates acoustic pressure of at least about 20 kPa.
 19. Themethod of claim 17, wherein said high power delivering of ultrasonicenergy generates acoustic pressure of at least about 50 kPa.
 20. Themethod of claim 17, wherein said high power delivering of ultrasonicenergy generates acoustic pressure of at least about 200 kPa.
 21. Themethod of claim 16, wherein said low frequency comprises a range ofabout 0.1 MHz to about 10 MHz.
 22. The method of claim 16, wherein saidlow frequency comprises a range of about 0.2 MHz to about 2 MHz.
 23. Themethod of claim 1, wherein said delivering of said therapeuticultrasonic energy is further adapted for: providing ultrasonic radiationforces for translating said microbubbles into or in the vicinity of thetreatment site.
 24. The method of claim 23, wherein said delivering ofsaid therapeutic ultrasonic energy for said translating comprises lowfrequency ultrasound transmission and reception.
 25. The method of 24,wherein said low frequency comprises a range of about 0.1 MHz to about10 MHz.
 26. The method of claim 24, wherein said low frequency comprisesa range of about 0.2 MHz to about 2 MHz.
 27. The method of claim 1,further comprising: delivering said microbubbles from an outlet portlocated on said catheter for translating said microbubbles into or inthe vicinity of the treatment site.
 28. The method of claim 1, furthercomprising: delivering said microbubbles from a port located on a secondcatheter or a lumen for translating said microbubbles into or in thevicinity of the treatment site.
 29. The method of claim 1, furthercomprising: delivering said microbubbles from a microfluidicflow-focusing device located on said catheter for translating saidmicrobubbles into or in the vicinity of the treatment site.
 30. Themethod of claim 1, further comprising: delivering said microbubbles froma microfluidic flow-focusing device located on a second catheter or alumen for translating said microbubbles into or in the vicinity of thetreatment site.
 31. The method of claim 1, wherein said microbubblescomprise a contrast agent.
 32. The method of claim 1, wherein saidmicrobubbles comprise a drug composition or agent composition, or drugand agent composition.
 33. The method of claim 32, wherein said drugcomposition or agent composition, or said drug and agent composition,being disposed: a) in shells of said microbubbles, b) in cores of saidmicrobubbles, c) outside said shells of said microbubbles, or d) anycombination of two or more of elements a, b and c.
 34. The method ofclaim 33, wherein said shells comprise lipids, phospholipids, oils,fats, lipopolymers, polymers, proteins, surfactants or combinationsthereof.
 35. The method of claim 33, wherein a thickness of said shellsmay vary from monomolecular 1 nm, to multimolecular and multilamellar,up to and including about 1,000 nm.
 36. The method of claim 33, whereina thickness of said shells may vary from monomolecular 0.1 nm, tomultimolecular and multilamellar, up to and including about 10,000 nm.37. The method of claim 32, wherein said drug composition comprises atleast one of the following: antiproliferative, immunosuppressive, orantiinflammatory drug.
 38. The method of claim 32, wherein said drugcomposition may be selected from the group consisting of: actinomycin-D,batimistat, c-myc antisense, dexamethasone, paclitaxel, taxanes,sirolimus, tacrolimus and everolimus, unfractionated heparin,low-molecular weight heparin, enoxaprin, bivalirudin, tyrosine kinaseinhibitors, Gleevec, wortmannin, PDGF inhibitors, AG1295, rho kinaseinhibitors, Y27632, calcium channel blockers, TRAM-34, IKCa channelblockers, amlodipine, nifedipine, and ACE inhibitors, S1P1 and/or S1P3receptor antagonists, sphingosine kinase 1 inhibitors, syntheticpolysaccharides, ticlopinin, dipyridamole, clopidogrel, fondaparinux,streptokinase, urokinase, r-urokinase, r-prourokinase, rt-PA, APSAC,TNK-rt-PA, reteplase, alteplase, monteplase, lanoplase, pamiteplase,staphylokinase, abciximab, tirofiban, orbofiban, xemilofiban,sibrafiban, roxifiban, an anti-restenosis agent, an anti-thrombogenicagent, an antibiotic, an anti-platelet agent, an anti-clotting agent, ananti-inflammatory agent, an anti-neoplastic agent, a chelating agent,penicillamine, triethylene tetramine dihydrochloride, EDTA, DMSA(succimer), deferoxamine mesylate, a radiocontrast agent, aradio-isotope, a prodrug, antibody fragments, antibodies, gene therapyagents, viral vectors and plasmid DNA vectors.
 39. The method of claim32, wherein said drug composition comprises a coated microbubble thatpossesses a selected molecular attachment ligand.
 40. The method ofclaim 1, wherein at least a plurality of said microbubbles comprise aninternal core filled with a gas, gas-vapor mixture or gas precursorphase, or any combination thereof.
 41. The method of claim 40, whereinsaid gas has a molecular mass from about 10 to about
 360. 42. The methodof claim 40, wherein said gas has a molecular mass from about 1 to about3,600.
 43. The method of claim 1, wherein said microbubbles comprise adecafluorobutane core.
 44. The method of claim 1, wherein at least aplurality of said microbubbles comprise shells, and said shells comprisea gas or mixture of two or more gases or gas precursors.
 45. The methodof claim 44, wherein said therapeutic ultrasonic energy is provided toat least one of the following: trigger transference into a gas phase,change volume of a gas core, or destroy the microbubbles.
 46. The methodof claim 1, wherein said delivering therapeutic ultrasonic energy andsaid delivering imaging ultrasonic energy comprise: activating anultrasound transducer source disposed within said distal region of saidcatheter.
 47. The method of claim 46, wherein said ultrasound transducersource comprises: a therapeutic transducer for delivering saidtherapeutic ultrasonic energy; and an imaging transducer for saidimaging of the treatment site.
 48. The method of claim 47, wherein saidtherapeutic transducer and said imaging transducer are essentiallylongitudinally aligned with one another.
 49. The method of claim 47,wherein said therapeutic transducer and said imaging transducer areessentially vertically overlaying one another.
 50. The method of claim46, wherein said ultrasound transducer source comprises: a) atherapeutic transducer for translating said microbubbles; b) an imagingtransducer for translating said microbubbles; c) a translatingtransducer for translating said microbubbles; d) any combination ofelements a, b or c; e) said ultrasound transducer source is configuredto perform any combination of a, b, or c; or f) said ultrasoundtransducer source is configured to perform any two of a, b, or c, andany one of a, b, or c.
 51. The method of claim 1, further comprising:gating said imaging ultrasonic energy and said therapeutic ultrasonicenergy, whereby they deliver energy temporally exclusively.
 52. Themethod of claim 1, further comprising: gating said imaging ultrasonicenergy and said therapeutic ultrasonic energy, whereby they deliverenergy simultaneously or at least partially simultaneously.
 53. Themethod of claim 1, further comprising: occluding or partially occludingsaid infusion of microbubbles upstream or downstream from the treatmentsite.
 54. The method of claim 1, further comprising: occluding orpartially occluding said infusion of microbubbles upstream from thetreatment site; and occluding or partially occluding said infusion ofmicrobubbles downstream from the treatment site.
 55. The method of claim1, wherein the treatment site is in a coronary artery.
 56. An ultrasoundcatheter system for providing therapy to a treatment site at one or morelocations of a subject, the system comprising: a tubular member having aproximal region and distal region and a lumen, said distal region ofsaid tubular member adapted to advance to or in proximity to thesubject's treatment site; a microbubble reservoir in or in hydrauliccommunication with said lumen of said tubular member, said microbubblereservoir is adapted to release microbubbles that are intended to belocated into or in proximity to the treatment site; a therapeuticultrasound transducer at said distal region of said tubular member, saidtherapeutic ultrasound transducer configured to deliver therapeuticultrasonic energy from within said distal region of said ultrasoundcatheter to said microbubbles to focally deliver a therapeutic materialto the treatment site; and an imaging ultrasound transducer at saiddistal region of said tubular member, said imaging ultrasound transducerconfigured to deliver imaging ultrasonic energy from within said tubularmember to the treatment site to provide real time imaging for guidingprecise delivery of said therapeutic material to the treatment site,wherein said imaging ultrasound transducer is mounted relative to saidtherapeutic ultrasound transducer so that the focal delivery of saidtherapeutic material is aligned with said real time imaging.
 57. Thesystem of claim 56 wherein said one or more locations comprise at leasta portion of an organ.
 58. The system of claim 57, wherein said organcomprises hollow organs, solid organs, parenchymal tissue, stromaltissue, and/or ducts.
 59. The system of claim 56, where the one or morelocations of the subject comprises at least a portion of a tubularstructure.
 60. The system of claim 59, wherein said tubular structurecomprises a blood vessel.
 61. The system of claim 56, wherein thesubject's treatment site comprises a vascular treatment site, and thevasculature treatment site comprises at least one of the following:stenosis region or any region exhibiting vascular disease.
 62. Thesystem of claim 56, wherein said imaging ultrasound transducer providesultrasonic energy for said imaging, and said ultrasonic energy for saidimaging comprises high frequency ultrasound transmission and reception.63. The system of claim 62, wherein said high frequency comprises arange of about 2 MHz to about 50 MHz.
 64. The system of claim 63,wherein said high frequency comprises a range of about 5 MHz to about 30MHz.
 65. The system of claim 62, wherein said high frequency comprises arange of about 12 MHz to about 50 MHz.
 66. The system of claim 62,wherein said high frequency comprises a range of about 12 MHz to about30 MHz.
 67. The system of claim 62, wherein said high frequencycomprises about 20 MHz.
 68. The system of claim 62, wherein said highfrequency comprises about 25 MHz.
 69. The system of claim 62, whereinsaid high frequency comprises about 30 MHz.
 70. The system of claim 56,wherein said therapeutic ultrasound transducer transmits and receiveslow frequency ultrasound energy.
 71. The system of claim 70, whereinsaid therapeutic ultrasound transducer provides ultrasonic energy foreffecting therapy, and said ultrasonic energy for said effecting therapycomprises high power.
 72. The system of claim 71, wherein high powerproduces pressure of at least about 20 kPa.
 73. The system of claim 71,wherein high power produces pressure of at least about 50 kPa.
 74. Thesystem of claim 71, wherein high power produces pressure of at leastabout 200 kPa.
 75. The system of claim 70, wherein said low frequencycomprises a range of about 0.1 MHz to about 10 MHz.
 76. The system ofclaim 70, wherein said low frequency comprises a range of about 0.2 MHzto about 2 MHz.
 77. The system of claim 56, wherein said therapeuticultrasound transducer provides therapeutic ultrasonic energy, and saidtherapeutic ultrasonic energy is further adapted for: providingultrasonic radiation forces for translating said microbubbles into or inthe vicinity of the treatment site.
 78. The system of claim 77, whereinsaid ultrasonic energy for said translating comprises low frequencyultrasound transmission and reception.
 79. The system of claim 78,wherein said low frequency comprises a range of about 0.1 MHz to about10 MHz.
 80. The system of claim 78, wherein said low frequency comprisesa range of about 0.2 MHz to about 2 MHz.
 81. The system of claim 56,further comprising: an outlet port disposed on said tubular member incommunication with said microbubble reservoir to allow microbubbles toexit from said reservoir, wherein said microbubbles being intended to betranslated into or in the vicinity of the treatment site.
 82. The systemof claim 56, further comprising: an outlet port disposed on a secondtubular member or a lumen in communication with said microbubblereservoir to allow microbubbles to exit from said reservoir, whereinsaid microbubbles being intended to be translated into or in thevicinity of the treatment site.
 83. The system of claim 56, furthercomprising: a microfluidic flow-focusing device in communication withsaid tubular member to allow microbubbles to exit from said reservoir,wherein said microbubbles being intended to be translated into or in thevicinity of the treatment site.
 84. The system of claim 56, furthercomprising: a microfluidic flow-focusing device in communication with asecond tubular member or a lumen to allow microbubbles to exit from saidreservoir, wherein said microbubbles being intended to be translatedinto or in the vicinity of the treatment site.
 85. The system of claim56, wherein said microbubbles comprises a contrast agent.
 86. The systemof claim 56, wherein said microbubbles comprise a drug composition oragent composition, or a drug and agent composition.
 87. The system ofclaim 86, wherein said drug or agent composition, or said drug and agentcomposition, being disposed: a) in shells of said microbubbles, b) incores or multiple cores of said microbubbles, c) outside said shells ofsaid microbubbles, or d) any combination of two or more of elements a, band c.
 88. The system of claim 87, wherein said shells comprise lipids,phospholipids, oils, fats, lipopolymers, polymers, proteins, surfactantsor combinations thereof.
 89. The system of claim 87, wherein a thicknessof said shells may vary from monomolecular 1 nm, to multimolecular andmultilamellar, up to and including about 1,000 nm.
 90. The system ofclaim 87, wherein a thickness of said shells-may vary from monomolecular0.1 nm, to multimolecular and multilamellar, up to and including about10,000 nm.
 91. The system of claim 86, wherein said drug compositioncomprises at least one of the following: antiproliferative,immunosuppressive, or antiinflammatory drug.
 92. The system of claim 86,wherein said drug composition may be selected from the group consistingof: actinomycin-D, batimistat, c-myc antisense, dexamethasone,paclitaxel, taxanes, sirolimus, tacrolimus and everolimus,unfractionated heparin, low-molecular weight heparin, enoxaprin,bivalirudin, tyrosine kinase inhibitors, Gleevec, wortmannin, PDGFinhibitors, AG1295, rho kinase inhibitors, Y27632, calcium channelblockers, TRAM-34, IKCa channel blockers, amlodipine, nifedipine, andACE inhibitors, S1P1 and/or S1P3 receptor antagonists, sphingosinekinase 1 inhibitors, synthetic polysaccharides, ticlopinin,dipyridamole, clopidogrel, fondaparinux, streptokinase, urokinase,r-urokinase, r-prourokinase, rt-PA, APSAC, TNK-rt-PA, reteplase,alteplase, monteplase, lanoplase, pamiteplase, staphylokinase,abciximab, tirofiban, orbofiban, xemilofiban, sibrafiban, roxifiban, ananti-restenosis agent, an anti-thrombogenic agent, an antibiotic, ananti-platelet agent, an anti-clotting agent, an anti-inflammatory agent,an anti-neoplastic agent, a chelating agent, penicillamine, triethylenetetramine dihydrochloride, EDTA, DMSA (succimer), deferoxamine mesylate,a radiocontrast agent, a radio-isotope, a prodrug, antibody fragments,antibodies, gene therapy agents, viral vectors and plasmid DNA vectors.93. The system of claim 86, wherein said drug composition comprising acoated microbubbles that posses a selected molecular attachment ligand.94. The system of claim 56, wherein a plurality of said microbubblescomprise an internal core filled with a gas, gas-vapor mixture or gasprecursor phase, or any combination thereof.
 95. The system of claim 94,wherein said gas has a molecular mass from about 10 to about
 360. 96.The system of claim 94, wherein said gas has a molecular mass from about1 to about 3,600.
 97. The system of claim 56, wherein said microbubblescomprise a decafluorobutane core.
 98. The system of claim 56, wherein atleast a plurality of said microbubbles comprise shells, said shellscomprising a gas or mixture of two or more gases or gas precursors. 99.The system of claim 98, wherein said therapeutic ultrasound transduceris configured to deliver therapeutic ultrasound energy to saidmicrobubbles to accomplish at least one of the following: triggertransference into a gas phase, change volume of a gas core, or destroythe microbubbles.
 100. The system of claim 56, wherein said an imagingplane of said imaging ultrasound transducer is coincident with at leastone of a point, line or plane of focus of said therapeutic ultrasonicenergy.
 101. The system of claim 100, wherein said imaging ultrasoundtransducer comprises an array of imaging transducers and saidtherapeutic ultrasound transducer comprises an array of therapeutictransducers.
 102. The system of claim 100, further comprising atranslating transducer for translating said microbubbles.
 103. Thesystem of claim 56, wherein said therapeutic ultrasound transducer andsaid imaging ultrasound transducer are essentially longitudinallyaligned with one another.
 104. The system of claim 56, wherein saidtherapeutic ultrasound transducer and said imaging ultrasound transducerare essentially vertically overlaying one another.
 105. The system ofclaim 56, wherein said control circuitry gates said imaging ultrasonicenergy and said therapeutic ultrasonic energy such that said imagingultrasonic energy said therapeutic ultrasonic energy are deliveredtemporarily exclusively.
 106. The system of claim 56, wherein saidcontrol circuitry gates said imaging ultrasonic energy and saidtherapeutic ultrasonic such that said imaging ultrasonic energy and saidtherapeutic ultrasonic energy are delivered-simultaneously or at leastpartially simultaneously.
 107. The system of claim 56, furthercomprising an occlusion device or partial-occlusion device configured tobe disposed either upstream or downstream from said treatment site toocclude or partially occlude said microbubbles.
 108. The system of claim56, further comprising: a first occlusion device or partial-occlusiondevice configured to be disposed upstream from said treatment site toocclude or partially occlude said microbubbles at said treatment site;and a second occlusion device or partial-occlusion device disposeddownstream from said treatment site to occlude or partially occlude saidinfused microbubbles at said treatment site.
 109. The system of claim56, wherein the treatment site is in a coronary artery.
 110. Anultrasound catheter system for providing therapy to a treatment site atone or more locations of a subject, the system comprising: a tubularmember having a proximal region and distal region and a lumen, saiddistal region of said tubular member adapted to advance to or inproximity to the subject's treatment site; a microbubble reservoir inhydraulic communication with said tubular member, said microbubblereservoir is adapted to release microbubbles that are intended to belocated into or in proximity to the treatment site; a therapeuticultrasound transducer at said distal region of said tubular member, saidtherapeutic ultrasound transducer configured to deliver therapeuticultrasonic energy from said distal region of said ultrasound catheter tosaid microbubbles to focally deliver a therapeutic material to thetreatment site; an imaging ultrasound transducer at said distal regionof said tubular member, said imaging ultrasound transducer configured todeliver imaging ultrasonic energy from said tubular member to thetreatment site to provide real time imaging for guiding precise deliveryof said therapeutic material to the treatment site, wherein said imagingultrasound transducer is mounted relative to said therapeutic ultrasoundtransducer so that the focal delivery of said therapeutic material isaligned with said real time imaging; and a port in a wall of saidtubular member, said port configured to permit passage of saidmicrobubbles from within said tubular member to a location outside ofsaid tubular member.
 111. The system of claim 110, wherein said port islocated proximally of said therapeutic ultrasound transducer.
 112. Thesystem of claim 110, wherein said microbubble reservoir is locatedinside of said tubular member.
 113. The system of claim 110, whereinsaid microbubble reservoir is in hydraulic communication with saidlumen.